Agents and methods related to reducing resistance to apoptosis-inducing death receptor agonists

ABSTRACT

Provided herein is a method of reversing or preventing a target cell&#39;s resistance to a death receptor agonist. Also provided are methods of screening for biomarkers resistance of and monitoring resistance to death receptor agonists. Also provided are methods of selectively inducing apoptosis in a target cell, treating a subject with cancer, autoimmune or inflammatory diseases, comprising administering compositions provided herein. Further provided are compositions comprising agents that modulate CARD containing proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of and claims priority toU.S. Ser. No. 11/814,551, filed Mar. 19, 2008, which is a §371 ofInternational Application No. PCT/US2006/03503 filed Jan. 31, 2006, nowexpired. International Application No. PCT/US2006/03503 claims priorityto U.S. Provisional Application No. 60/649,437, filed Feb. 2, 2005. Theapplications which the present application claims priority to are hereinincorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos.P50CA83591, P50CA89019, and U19AI056542 awarded by the NationalInstitutes of Health. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The disclosed invention relates generally to agents that inhibitresistance to apoptosis-inducing agonists of death receptors and the useof such agents and agonists and biomarkers in the treatment of cancerand autoimmune or inflammatory diseases.

BACKGROUND OF THE INVENTION

TNF-related apoptosis-inducing ligand (TRAIL), a member of the TNFsuperfamily, has a strong apoptosis-inducing activity against cancercells (Wiley, S. R., et al. 1995. Immunity 3:673-682). Unlike otherdeath-inducing ligands of the TNF superfamily such as TNF-α and Fasligand, TRAIL has been of particular interest in the development ofcancer therapeutics as it preferentially induces apoptosis of tumorcells, having little or no effect on normal cells (Walczak, H., et al.1999. Nat Med 5:157-163). At least five receptors for TRAIL have beenidentified, two of which, DR4 (TRAIL-R1) and DR5 (TRAIL-R2), are capableof transducing the apoptosis signal (Walczak, H., et al. 1997. Embo J16:5386-5397; Pan, G., et al. 1997. Science 276:111-113; Chaudhary, P.M., et al. 1997. Immunity 7:821-830) whereas the other three (TRAIL-R3,—R4 and OPG) serve as decoy receptors to block TRAIL-mediated apoptosis(Pan, G., et al. 1997. Science 277:815-818; Marsters, S. A., et al.1997. Curr Biol 7:1003-1006; Emery, J. G., et al. 1998. J Biol Chem273:14363-14367). Like Fas and TNFR1, the intracellular segments of bothDR4 and DR5 contain a death domain and transduce an apoptosis signalthrough a FADD- and caspase 8-dependent pathway (Walczak, H., et al.1997. Embo J 16:5386-5397; Chaudhary, P. M., et al. 1997. Immunity7:821-830; Kuang, A. A., et al. 2000. J Biol Chem 275:25065-25068).Administration of the recombinant soluble form of TRAIL in experimentalanimals, including mice and primates, induces significant tumorregression without systemic toxicity (Walczak, H., et al. 1999. Nat Med5:157-163). However, as TRAIL has been shown to elicit side effects suchas liver toxicity in humans, other agonists of TRAIL receptors have beendeveloped.

Selective targeting of DR5 with a unique agonistic monoclonal anti-DR5antibody, TRA-8, and its humanized or human versions thereof caneffectively and selectively induce apoptosis of tumor cells. AllTRAIL-sensitive cancer cells have been found to be susceptible toTRA-8-mediated apoptosis. Chemotherapeutic agents can synergisticallyenhance TRAIL-mediated apoptosis of tumor cells both in vitro and invivo. For example, the combination therapy of TRA-8 with Adriamycinresulted in a significantly higher complete tumor regression rate thaneither agent alone (Buchsbaum, D. J., et al. 2003. Clin Cancer Res9:3731-3741). These results suggest that chemotherapeutic agents mightregulate the signal transduction of DR5 or the threshold of signalingrequired to induce apoptosis. TRA-8 has been selected as a candidate fordevelopment as a cancer therapy based on its efficacy and safety.Pre-clinical studies indicate that TRA-8 has a very strong anti-cancerefficacy in xenograft models of human cancer, particularly incombination with chemotherapy (Buchsbaum, D. J., et al. 2003. ClinCancer Res 9:3731-3741). There is further indication that monkeystolerate systemic administration of TRA-8 well. The binding of TRA-8 tomonkey DR5 is similar to that of human DR5, and the monkeys tolerateddoses as high as 48 mg/kg dose.

The expression of a death receptor by a target cell, however, is notnecessarily sufficient to make the cell susceptible to the induction ofapoptois by a ligand for the receptor. As an example, although mostcancer cells express high levels of DR5, they are not necessarilysusceptible to apoptosis induced by TRA-8, which is specific for DR5 anddoes not react with the decoy receptors. Furthermore, target cells suchas cancer cells can show resistance to TRA-8 or other agents that induceapoptosis through death receptors (e.g., DR4 or DR5). Needed in the artis a biomarker to predict resistance and a means of reducing resistanceof target cells to agonists of death receptors such as TRA-8.

BRIEF SUMMARY OF THE INVENTION

In accordance with the purpose(s) of this invention, as embodied andbroadly described herein, this invention, in one aspect, relates to amethod of reversing or preventing a target cell's resistance to a deathreceptor agonist comprising contacting the target cell with a modulatorof one or more activities of a CARD containing protein, wherein themodulation reverses or prevents resistance to the agonist.

Provided herein is a method of screening a cell for a biomarker ofresistance to a death receptor agonist comprising assaying the cell fortotal DDX3, or a homologue thereof, wherein high levels signifyresistance to the agonist.

Provided herein is a method of screening a cell for a biomarker ofresistance to a death receptor agonist comprising assaying theassociation of the death receptor and a CARD containing protein, whereinhigh levels of association signify resistance to the agonist.

Provided herein is a method of screening a cell for a biomarker ofresistance to a death receptor agonist comprising a) contacting the cellwith the death receptor agonist, b) monitoring the fractionalassociation of the death receptor and a CARD containing protein, whereinassociation signifies resistance to the agonist.

Further provided is a method of screening a cell for a biomarker ofresistance to a death receptor agonist comprising monitoring theassociation of a caspase or modulator of caspases (eg, cIAP1, cIAP2,XIAP, survivin) with the CARD containing protein and comparing the levelof association with a sample from known resistant and non-resistantcontrol cells, wherein the association of IAPs with the CARD containingprotein at levels similar to that of resistant cells signifiesresistance to the agonist. Optionally, the cell to be screened ispre-contacted with a death receptor agonist (e.g. agonistic antibody).

Provided herein is a method of monitoring resistance to a death receptoragonist in a subject, comprising (a) acquiring a biological sample fromthe subject and (b) detecting association of a CARD containing proteinwith a death receptor in the sample, the association indicatingresistance.

Further provided is a method of monitoring resistance to a deathreceptor agonist in a subject, comprising (a) acquiring a biologicalsample from the subject and (b) detecting association of a caspase ormodulator of caspase with a CARD containing protein in the sample, theassociation indicating resistance.

Also provided is a method of selectively inducing apoptosis in a targetcell expressing a death receptor, comprising the steps of (a) contactingthe target cell with a therapeutic amount of a death receptor agonistthat specifically binds the death receptor and (b) administering to thetarget cell a therapeutic amount of a modulator of one or moreactivities of a CARD containing protein.

Provided is a method of treating a subject with cancer, comprisingadministering to the subject a therapeutic amount of (a) a deathreceptor agonist and (b) a modulator of one or more activities of a CARDcontaining protein, wherein the modulator reduces resistance to thedeath receptor agonist.

Also provided is a method of treating a subject with an inflammatory orautoimmune disease, comprising administered to the subject a therapeuticamount of (a) a death receptor agonist and (b) an agent that modulatesone or more activities of a CARD containing protein, wherein themodulator reduces resistance to the death receptor agonist.

Provided herein is a composition comprising (a) a death receptor agonistand (b) an agent that modulates one or more activities of a CARDcontaining protein, wherein the modulator reduces resistance to thedeath receptor agonist.

Further provided is an isolated nucleic acid comprising an shRNA,wherein the shRNA inhibits the expression of a CARD containing protein.

Also provided is an isolated polypeptide encoding the CARD containingprotein binding region of a death receptor, wherein the polypeptidecomprises fewer than 25 amino acid residues.

Further provided is an isolated polypeptide comprising the deathreceptor binding domain of a CARD containing protein.

Additional advantages of the disclosed method and compositions will beset forth in part in the description which follows, and in part will beunderstood from the description, or may be learned by practice of thedisclosed method and compositions. The advantages of the disclosedmethod and compositions will be realized and attained by means of theelements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of thedisclosed method and compositions and together with the description,serve to explain the principles of the disclosed method andcompositions.

FIG. 1 shows induction of tumor cell resistance to TRA-8-mediatedapoptosis. Panel (A) shows flow cytometry analysis of cell surfaceexpression of TRAIL-R1 and TRAIL-R2. Human breast cancer cell line,MDA-231 and human ovarian cancer cell lines, UL-3C were stained withCyChrome-conjugated anti-TRAIL-R1 (2E12) and PE-conjugated anti-TRAIL-R2(2B4), and analyzed by FACScan flow cytometer. Panel (B) showssusceptibility of MDA-231 and UL-3C cells to TRA-8, 2E12 orTRAIL-mediated apoptosis. Cells were cultured in 96-well plate with1,000 cells per well in triplicates and incubated with indicatedconcentrations of each apoptosis-inducing agent. For 2E12-inducedapoptosis, 2 μg/ml goat anti-mouse IgG1 was added, and for TRAIL-inducedapoptosis, anti-Flag antibody was added as the crosslinker. Cellviability was determined after overnight culture by ATPLITE assay. Cellviability was determined by the percentage of the counts of the treatedwells over that of medium control. Each point represents an average oftriplicates, and is representative for at least three independentexperiments. Panel (C) shows susceptibility of tumor cells toTRA-8-mediated apoptosis during induction of TRA-8 resistance. Inductionof TRA-8 resistance was initiated by the treatment of cells with 1 ng/mlof TRA-8 for two days. The TRA-8 doses were doubled every two days until2,000 ng/ml. At each cycle of the doses, cell viability of non-inducedand induced cells under the treatment with each correspondent dose wasdetermined by ATPLITE assay. Data are presented as an average of thetriplicate culture.

FIG. 2 shows selectivity of induced TRA-8 resistance in MDA-231 andUL-3C cells. Panel (A) shows TRAIL-R2-induced apoptosis in MDA-231 andUL-3C cells. Both parental and resistant MDA231 and UL-3C cells weretreated with indicated concentrations of TRA-8. Panel (B) showsTRAIL-R1-induced apoptosis in MDA-231 and UL-3C cells. Both parental andresistant MDA231 and UL-3C cells were treated with indicatedconcentrations of 2E12. Panel (C) shows TRAIL-induced apoptosis inMDA-231 and UL-3C cells. Both parental and resistant MDA231 and UL-3Ccells were treated with indicated concentrations of recombinant solubleTRAIL. Cell viability was determined after overnight culture by ATPLITEassay as described above. Panel (D) shows maintenance of TRA-8resistance. After induction of TRA-8 resistance, TRA-8 was withdrawn.The maintenance of TRA-8 resistance was determined every week afterwithdraw of TRA-8. Cells were treated with 1,000 ng/ml TRA-8 overnight,and cell viability was determined by ATPLITE assay.

FIG. 3 shows TRAIL-R2 and associated apoptosis regulatory expression inTRA-8 resistant cells. Panel (A) shows shows Western blot analysis ofprotein expression. Total cell lysates were separated in SDS-PAGE andWestern blotted. The blots were probed with 1 μg/ml primary antibodyovernight and followed by HRP-conjugated secondary antibody. Theproteins were revealed by ECL chemiluminescence. Panel (B) shows cDNAarray analysis of MDA231 parental and resistant cells. The membrane cDNAarrays for a panel of human apoptosis (upper panel) and cell signalingassociated genes (lower panel) were purchased from SuperArray, Inc. The³²P labeled cDNA probes were prepared from total RNA of MDA231 parentaland resistant cells and hybridized with the cDNA array on the blot. Thegene expression profiles were analyzed with CyClone Phosphor-Imager.

FIG. 4 shows activation of caspase pathway and JNK/p38 kinase pathway inTRA-8 resistant cells. Panel (A) shows TRAIL-R1 and -R2-triggeredcaspase activation. MDA231 parental and resistant cells were treatedwith 1,000 ng/ml TRA-8 (left panel) or 2E12 (right panel) for indicatedtime. Western blot of total cell lysates were probed with polyclonalanti-caspase 8 (upper panel), anti-caspase 3 (middle panel) or anti-PARP(lower panel). The arrows indicate the full-length and cleaved proteins.Panel (B) shows activation of JUK/p38 kinase pathway. Cells were treatedin the same way as described above. Western blots of total cell lysateswere probed with polyclonal anti-phosphorylated JNK (upper panel) oranti-phosphorylated p38 (lower panel). The arrows indicate thephosphorylated proteins.

FIG. 5 shows altered DISC formation in TRA-8 resistant cells. Panel (A)shows co-immunoprecipitation assay of DISC formation. MDA231 parentaland resistant cells were treated with 1,000 ng/ml TRA-8 (left panel) or2E12 (right panel) for indicated time. TRAIL-R1 and TRAIL-R2 wereimmunoprecipitated with 2E12 or 2B4-conjugated Sepharose 4B. Westernblots of the co-immunoprecipitated proteins and total cell proteins wereprobed with polyclonal anti-FADD (upper panel) or anti-caspase 8antibody (middle panel) or anti-cFLIP antibody (lower panel). Panels(B-E) show two dimension proteomic profile of TRAIL-R2-associatedproteins of TRA-8 resistant cells. MDA231 parental and resistant cellswere treated with 1,000 ng/ml TRA-8 for four hours or remained untreatedas control. After TRAIL-R2 immunoprecipitation with 2B4-conjugatedSepharose 4B, the eluted proteins were separated by two dimensionelectrophoresis and stained with the SYPRO Ruby staining buffer. Thedifferentially expressed protein spots as circled were identified by thePDQuest software. The experiments were repeated at least three times fora reproducible result.

FIG. 6 show reversal of TRA-8 resistance by chemotherapeutic agents.Panel (A) shows susceptibility of TRA-8 resistant cells to TRA-8-induceapoptosis in the presence of chemotherapeutic agents. MDA231 and UL-3Cresistant cells were treated with variable doses of TRA-8 in the absenceor presence of 0.1 CM taxol, 1 VM Adriamycin, 100 VM cisplatin or 5 VMBisVIII. Cell viability was determined after overnight culture byATPLITE assay. Panel (B) shows activation of caspase cascade in TRA-8resistant cells by Adriamycin. MDA231 resistant cells were culture inmedium control (lane) or treated with 1,000 ng/ml TRA-8 and 1 MAdriamycin for one hour (lane 2) or four hours (lane 3), or 1 VMAdriamycin alone or 1,000 ng/ml TRA-8 alone (lane 5). Western blots oftotal cell lysates were probed with monoclonal anti-human caspaseantibodies. Panel (C) shows TRAIL-R2 recruitment of FADD in TRA-8resistant cells. TRAIL-R2 from differently treated MDA231 resistantcells as indicated above was immunoprecipitated with 2B4 sepharose 4B.Co-immunoprecipitation of FADD with TRAIL-R2 was determined by Westernblot probed with anti-FADD antibody.

FIG. 7 shows DDX3 association with TRAIL-R2. MDA231 parental cells andresistant cells were transfected with the recombinant full-length DDX3.48 hours after transfection, cells were treated with 500 ng/ml TRA-8 forthe indicated time. Total cell proteins were probed with monoclonalanti-His antibody (upper panel). β-actin was used as the loadingcontrol. Co-immunoprecipitation assay of recombinant DDX3 associatedwith TRAIL-R2 determined using anti-His antibody (lower panel). Toanalyze endogenous DDX3 associated with TRAIL-, MDA231 parental cellsand resistant cells were treated with 500 ng/ml TRA-8 for the indicatedtime. TRAIL-R2 was immunoprecipitated with 2B4-conjugated Sepharose 4B.Total cell proteins were probed with monoclonal anti-DDX3 antibodies,3E2 and 5A6 (upper panel). The co-immunoprecipitated endogenous DDX3 wasprobed with monoclonal anti-DDX3 antibodies, 3E2 and 5A6 (lower panel).TRAIL-R2 in parental cells and resistant cells were determined byWestern Blot with anti-TRAIL-R2 polyclonal antibody.

FIG. 8 shows mapping of the interaction region of DDX3 and TRAIL-R2.FIG. 8A shows constructs of deleted DDX3. The cDNAs encoding thefull-length and deleted DDX3 as indicated were cloned into pcDNA3.1-HisAexpression vector. 293 cells were transfected with the N-terminal,C-terminal deleted DDX3, and wild-type DDX3. 48 hours aftertransfection, the recombinant DDX3 expressions were detected by Westernblot using anti-His antibody (upper panel).TRAIL-R2-co-immunoprecipitated recombinant DDX3 were determined byWestern blot analysis using anti-His monoclonal antibody (middle panel).TRAIL-R2 were determined by Western Blot with anti-TRAIL-R2 polyclonalantibody (lower panel). Lane 1: non-transfection. Lane 2-5: Δ2-5 ofN-terminal DDX3. Lane 6-9: Δ3-6 of C-terminal DDX3. Lane 10: thefull-length DDX3. FIG. 8B shows the interaction of DDX3 with TRAIL-R2 isindependent on death domain. The cDNAs encoding the full-length anddeleted TRAIL-R2 as indicated were cloned into a shuttle-CMV vector.Murine 3T3 cells were co-transfected with either wild-type or mutantTRAIL-R2 and DDX3. 24 hours later, cell surface expression was examinedby flow cytometry analysis using TRA-8 and PE-conjugated anti-mIgG1. 48hours after co-transfection, cell lysates were immunoprecipitated withTRA-8. Total DDX3 (upper panel) and TRAIL-R2 associated DDX3 (middlepanel) were examined by Western blot using anti-His antibody. Lane 1:non-transfection; Lane 2: DDX3 alone; lane 3: wild-type TRAIL-R2 andDDX3; lane 4-9: Δ1-6 of TRAIL-R2 and DDX3. TRAIL-R2 were determined byWestern Blot with anti-TRAIL-R2 polyclonal antibody (lower panel). FIG.8C shows the cDNAs encoding the full-length and truncated TRAIL-R2 asindicated were cloned into a dual-promoter expression vector with GFP asa reporter protein. Murine 3T3 cells were co-transfected with eitherwild-type or mutant TRAIL-R2 and DDX3. 24 hours later, cell surfaceexpression was examined by flow cytometry analysis using TRA-8 andPE-conjugated anti-mIgG1. 48 hours after co-transfection, cell lysateswere immunoprecipitated with TRA-8. TRAIL-R2 were determined by WesternBlot with anti-TRAIL-R2 polyclonal antibody (upper panel). Total DDX3(lower panel) and TRAIL-R2 associated DDX3 (middle panel) were examinedby Western blot using anti-His antibody. Lane 1: DDX3 alone; lane 2:ΔTRAIL-R2-300-330 and DDX3; lane 3: ΔD330 and DDX3; lane 4: ΔD340 andDDX3; lane 5: wild-type TRAIL-R2 and DDX3. FIG. 8D shows amino acidalignment of the DDX3 binding region of TRAIL-R2 with DcR2 and DR4. FIG.8E shows DDX3 serves as the link between TRAIL-R2 and cIAP1. MDA231parental cells and MDA231-resistant cells were treated with 500 ng/mlTRA-8 for the indicated time. TRAIL-R2 was immunoprecipitated with2B4-conjugated Sepharose 4B. DDX3 was immunoprecipitated with3E4-conjugated Sepharose 4B. Western blots of the total DDX3, cIAP1(upper panel), TRAIL-R2 co-immunoprecipitated DDX3, cIAP1 (middlepanel), DDX3 co-immunoprecipitated DDX3, cIAP1 (lower panel) were probedwith 3E4, monoclonal anti-DDX3 antibody, and 1C12, monoclonal anti-cIAP1antibody. TRAIL-R2 was determined by Western Blot using anti-TRAIL-R2polyclonal antibody (middle panel).

FIG. 9 shows Down-regulation of DDX3 reverses resistance toTRA-8-induced apoptosis. FIG. 9A shows selected effective sRNAi-DDX3.MDA231 parental cells were transfected with U6-Entry vector encodingtargets sRNAi-DDX3. 48 hours after transfection, DDX3 expression wasdetermined by Western blot analysis using anti-DDX3 antibody. β-actinwas used as the loading control. FIG. 9B shows MDA231-resistant cellswere co-transfected with GFP expression vector and sRNAi-DDX3. 24 hoursafter transfection, GFP-positive cells were sorted by cytometry andcultured with various concentrations of TRA-8 overnight. DDX3expressions were detected by Western blot using anti-DDX3 antibody(upper panel). TRAIL-R2 was immunoprecipitated with 2B4-conjugatedSepharose 4B. TRAIL-R2 associated DDX3 were probed with 3E4, monoclonalanti-DDX3 antibody (middle panel). 24 hours after transfection, thecells were treated with various concentrations of TRA-8 overnight. Thesusceptibility of transfected cells to TRA-8-induced apoptosis wasdetermined by ATPLite assay. FIG. 9D shows the transfected cellsundergoing apoptosis were determined by TUNEL staining. FIG. 9E showsthe panels of cancer cells were transfected with control or DDX3 sRNAioligo. 48 hours after transfection, reduced expression of DDX3 wasdetected by Western blot using anti-DDX3 antibody. β-actin was used asthe loading control. FIG. 9F shows 24 hours after transfection, cellswere treated with various concentrations of TRA-8 overnight. Thesusceptibility of transfected cells to TRA-8-induced apoptosis wasdetermined by ATPLite assay.

FIG. 10 shows TRAIL-R2 lacking DDX3 binding motif is pro-apoptotic.Murine 3T3 cells were co-transfected with ΔD340-TRAIL-R2 and DDX3,wild-type TRAIL-R2 and DDX3, ΔT300-330-TRAIL-R2 and DDX3. 24 hours aftertransfection, cells were treated with 500 ng/ml TRA-8 overnight.Apoptotic cells were determined by PE-conjugated anti-TRAIL-R2 antibody,biotin-conjugated annexin V in GFP-positive cells using flow cytometryanalysis. Apoptotic cells were shown by the column bar graph.

FIG. 11 shows DDX3 serves as adaptor protein linking cIAP1 to TRAIL-R2.FIG. 11A shows mapping of cIAP1 binding CARD of DDX3. 293 cells weretransfected with a series of deleted DDX3 as indicated (upper panel),the N-terminal aa 1-aa 50 deleted (lane 1), the N-terminal aa 1-aa 100deleted (lane 2), the N-terminal aa 1-aa 150 deleted (lane 3) andC-terminal aa 350-aa 662 deleted DDX3 (lane 4) and the full-length (lane5). 48 hours after transfection, the recombinant DDX3 wasimmunoprecipitated with the nickel beads. Total cIAP1 (middle panel) andco-immunoprecipitated cIAP1 were determined by Western blot analysisusing anti-cIAP1 monoclonal antibody (lower panel). FIG. 11B shows DDX3lacking CARD reverses TRA-8 resistance. Four lines of TRA-8-resistanttumor cells were transfected with the adenoviral vector encoding thefull-length DDX3 (DDX3-FL) or the CARD-truncated DDX3 (ΔCARD-DDX3). 48hours after transfection, cell lysates were immunoprecipitated withTRA-8 and followed Western blot analysis of co-immunoprecipitated DDX3(upper panel), and cIAP1 (middle panel). TRAIL-R2 were determined byWestern Blot using anti-TRAIL-R2 polyclonal antibody (lower panel). FIG.11C shows cell viability determined by ATPLite assay. The transfectedcells were incubated with 500 ng/ml TRA-8 overnight.

FIG. 12 shows TRAIL-R2/DDX3/cIAP1 protein complexes block caspase-8activation and DDX3 cleavage in TRA-8-resistant cells. FIG. 12 A showsTRAIL-R2/DDX3/cIAP1 protein complexes in TRA-8-sensitive and -resistantcells. MDA231 parental (MDA231p) and induced resistant (MDA231r) cells,UL-3C parental (UL-3 Cp) and induced resistant (UL-3Cr) cells weretreated with 500 ng/ml TRA-8 for eight hours. Total cell lysates wereimmunoprecipitated with 2B4 anti-TRAIL-R2 antibody-conjugated sepharose4B and eluted with glycine-HCl pH2.0 and immediately neutralized with 1MTris buffer. ELISA plates were coated with 2B4 anti-TRAIL-R2 antibodyand blocked with 3% BSA PBS. After incubation with the protein complexfrom the TRAIL-R2 co-IP, TRAIL-R2 was detected by biotin-conjugatedpolyclonal anti-TRAIL-R2 antibody, followed by HRP-conjugatedstreptavidin. FIG. 12B shows DDX3 was detected by biotin-conjugated 3E2,anti-DDX3 antibody, followed by HRP-conjugated streptavidin. FIG. 12Cshows cIAP1 was detected by biotin-conjugated 1C12, anti-cIAP1 antibody,followed by HRP-conjugated streptavidin. FIG. 12D shows differentialsusceptibility of DDX3 to caspase-mediated cleavage. DDX3 was isolatedfrom indicated cells by immunoprecipitation with 2B4 anti-TRAIL-R2antibody-conjugated sepharose 4B, and incubated with indicated caspase-2or caspase-8 at 37° C. from 4 hours. FIG. 12E shows the amount of DDX3was measured by sandwich ELISA using 5A6 as capture and 3E2 as detectionantibody. The results are presented as percentage of DDX3 after cleavageover non-cleaved controls. FIG. 12F shows effect of DDX3/cIAP1 complexon caspase 8 activity. DDX3 was isolated from the indicated cells by byimmunoprecipitation with 2B4 anti-TRAIL-R2 antibody-conjugated sepharose4B, and incubated with recombinant active human caspase-8 in thepresence of a fluorescent substrate of caspase-8, Ac-IETD-AMC, for 2hours. The inhibition of caspase 8 was measured by decreasedfluorescence intensity. The results are presented as percentage ofmaximum activity in control wells.

FIG. 13 shows the role of the serine-rich domain of DDX3 in regulationof the association and cleavage. Panel (A) shows a conserved domain forGSK3 substrate. Panel (B) shows DDX3 co-immunoprecipitated by GSK3α(top). TRA-8 sensitive MDA231 cells were treated with TRA-8 with orwithout lithium for two hours. Total cell lysate was immunoprecipitatedwith anti-GSK3α beads. The proteins with GSK3α were analyzed by Westernblot using anti-DDX3 antibody. GSK3 phosphorylates DDX3 (B, lower). Therecombinant DDX3 and tau were used as the substrates incubated with GSKwith or without PKA for one hour. The incorporated 32P was counted andpresented as cpm. (C) GSK3 fails to phosphorylate Ser90 mutant DDX3. (D)MDA231 cells were transfected with wild-type DDX3 and Ser90 mutant DDX3.After TRA-8 treatment, the disassociation of DDX3 from DR5 and cleavageof DDX3 were determined.

DETAILED DESCRIPTION OF THE INVENTION

The disclosed method and compositions may be understood more readily byreference to the following detailed description of particularembodiments and the Example included therein and to the Figures andtheir previous and following description.

The induction of death receptor-mediated apoptosis of tumor cells is anextremely promising approach for cancer therapy. As in most, if not all,therapies, some target cells are resistant. As an example, TRA-8, aunique agonistic monoclonal anti-DR5 antibody, induces apoptosis ofhuman cancer cells without hepatocyte cytotoxicity (Ichikawa, K., et al.2001. Nat Med 7:954-960), exhibits strong anti-cancer efficacy in animalmodels (Buchsbaum, D. J., et al. 2003. Clin Cancer Res 9:3731-3741), andhas demonstrated safety in toxicity studies in non-human primates. Thus,TRA-8 is used as an example herein but other agents that induceapoptosis through death receptor (e.g., DR4 or DR5) activation can be beused in the methods taught herein. While TRA-8 and its humanized andhuman versions are under clinical development as an anti-cancer therapy,some tumor cell lines are resistant to TRA-8-mediated apoptosis despitereasonable levels of DR5 expression. These observations suggest that theresistance is not related to receptor expression but rather toDR5-initiated signaling mechanisms. Certainly DR5-mediated apoptosis canbe enhanced significantly by common chemotherapeutic agents (Ohtsuka,T., and T. Zhou. 2002. J Biol Chem 277:29294-29303; Ohtsuka, T., D. etal. 2003. Oncogene 22:2034-2044). Disclosed are compositions and methodsto inhibit resistance to death receptor agonists by targeting a familyof CARD containing proteins that bind death receptors and inhibitcaspase activation.

It is to be understood that the disclosed methods and compositions arenot limited to specific synthetic methods, specific analyticaltechniques, or to particular reagents unless otherwise specified, and,as such, may vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only andis not intended to be limiting.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials aredisclosed that while specific reference of each various individual andcollective combinations and permutation of these compounds may not beexplicitly disclosed, each is specifically contemplated and describedherein. For example, if a vector is disclosed and discussed and a numberof vector components including the promoters are discussed, each andevery combination and permutation of promoters and other vectorcomponents and the modifications that are possible are specificallycontemplated unless specifically indicated to the contrary. Thus, if aclass of molecules A, B, and C are disclosed as well as a class ofmolecules D, E, and F and an example of a combination molecule, A-D isdisclosed, then even if each is not individually recited, each isindividually and collectively contemplated. Thus, is this example, eachof the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F arespecifically contemplated and should be considered disclosed fromdisclosure of A, B, and C; D, E, and F; and the example combination A-D.Likewise, any subset or combination of these is also specificallycontemplated and disclosed. Thus, for example, the sub-group of A-E,B-F, and C-E are specifically contemplated and should be considereddisclosed from disclosure of A, B, and C; D, E, and F; and the examplecombination A-D. This concept applies to all aspects of this applicationincluding, but not limited to, steps in methods of making and using thedisclosed compositions. Thus, if there are a variety of additional stepsthat can be performed it is understood that each of these additionalsteps can be performed with any specific embodiment or combination ofembodiments of the disclosed methods, and that each such combination isspecifically contemplated and should be considered disclosed.

A variety of sequences are provided herein and these and others can befound in Genbank, at ‘www.pubmed.gov’. Those of skill in the artunderstand how to resolve sequence discrepancies and differences and toadjust the compositions and methods relating to a particular sequence toother related sequences. Primers and/or probes can be designed for anysequence given the information disclosed herein and known in the art.

Provided is a method of reversing or preventing a target cell'sresistance to a death receptor agonist comprising contacting the targetcell with a modulator of one or more activities of a CARD containingproteins, wherein the modulation reverses or prevents resistance to theagonist. The method has utility for apoptosis signaling research andtherapeutic treatment of diseases such as cancer and autoimmune andinflammatory disorders. Thus, the contacting step of the method can beperformed in vivo or in vitro.

As used throughout, “reverse” or “reversing” means to change to theopposite position, direction, or course, such as in to change the courseof a disease from that of getting worse to that of getting better. Forexample, in the case of death receptor resistance, to reverse a targetcell's resistance to a death receptor agonist is to make the cell lessresistant to said agonist. Thus, for example, reversing the resistanceof a target cell that is 100% resistant can result in said target cellbeing 90% to 0% resistant to the death receptor agonist, including 90%,85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%,15%, 10%, 5%, and 0% resistant.

As used throughout, “preventing” means to preclude, avert, obviate,forestall, stop, or hinder something from happening, especially byadvance planning or action. For example, in the case of death receptorresistance, to prevent a target cell's resistance to a death receptoragonist is to make the cell less capable of becoming resistant to saidagonist. Thus, for example, preventing 100% resistance in a target cellcan result in said target cell being only 0% to 90% resistant to thedeath receptor agonist, including 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, and 90% resistant.

“Reversing” or “preventing” refers to a change in magnitude or a delayin any change in magnitude. Thus, in the case of death receptorresistance, “reversing” or “preventing” includes reducing the course ofincreasing resistance or delaying an increase in resistance.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “apolypeptide” includes a plurality of such polypeptides, reference to“the polypeptide” is a reference to one or more polypeptides andequivalents thereof known to those skilled in the art, and so forth.

“Optional” or “optionally” means that the subsequently described event,circumstance, or material may or may not occur or be present, and thatthe description includes instances where the event, circumstance, ormaterial occurs or is present and instances where it does not occur oris not present.

Ranges may be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, also specifically contemplated and considered disclosed isthe range from the one particular value and/or to the other particularvalue unless the context specifically indicates otherwise. Similarly,when values are expressed as approximations, by use of the antecedent“about,” it will be understood that the particular value forms another,specifically contemplated embodiment that should be considered disclosedunless the context specifically indicates otherwise. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint unless the context specifically indicates otherwise. Finally,it should be understood that all of the individual values and sub-rangesof values contained within an explicitly disclosed range are alsospecifically contemplated and should be considered disclosed unless thecontext specifically indicates otherwise. The foregoing appliesregardless of whether in particular cases some or all of theseembodiments are explicitly disclosed.

As used throughout, “target cell” means a cell bearing the targeteddeath receptor, including, for example, a cell that expresses DR5 or DR4and illustratively includes abnormally growing cells and tumor cellssuch as papillomas and warts; breast cancer, colon cancer, hepatomas,leukemias, lung cancer, melanoma, myelomas, osteosarcomas, ovariancancer, pancreatic cancer, prostate cancer, cancer of the head and neck,thyroid cancer, uterine cancer, tumors of the brain such asastrocytomas, activated immune cells (e.g., activated lymphocytes,lymphoid and myeloid cells), inflammatory cells, rheumatoid arthritissynovial cells, and virally infected cells. In vivo, the target cell isa cell of an individual with a pathological condition, including thosein which cell proliferation is abnormal or dysregulated such as cancerand rheumatoid arthritis. Target cells include human, non-human primate,cats, dogs, rat, mouse, guinea pig, rabbit, goat, sheep, cow, horse,chicken, pig, marmoset and ferret cells, or cells of cells of variouscell lines (e.g., Jurkat cells).

By “death receptor” is meant a receptor that induces cellular apoptosisonce bound by a ligand. Death receptors include, for example, tumornecrosis factor (TNF) receptor superfamily members having death domains(e.g., TNFRI, Fas, DR3, 4, 5, 6) and TNF receptor superfamily memberswithout death domains LTbetaR, CD40, CD27, HVEM.

Signal transduction through, for example, DR5 is a key mechanism in thecontrol of DR5-mediated apoptosis. A common feature of the deathreceptors of the TNFR superfamily is that they all have a conserved“death domain” in their cytoplasm tail (Zhou, T., et al. 2002. ImmunolRes 26:323-336). It is well established that DR5-mediated apoptosis isinitiated at the death domain. Crosslinking of DR5 at cell surface byTRAIL or agonistic anti-DR5 antibody leads to oligomerization of DR5,which is immediately followed by the recruitment of FADD to the deathdomain of DR5 (Bodmer, J. L., et al. 2000. Nat Cell Biol 2:241-243;Chaudhary, P. M., et al. 1997. Immunity 7:821-830; Kuang, A. A., et al.2000. J Biol Chem 275:25065-25068; Schneider, P., et al. 1997. Immunity7:831-836; Sprick, M. R., et al. 2000 Immunity 12:599-609). Thedeath-domain engaged FADD further recruits the initiator procaspase 8and/or procaspase 10 to form a DISC through homophilic DD interactions(Krammer, P. H. 2000. Nature 407:789-795). The activated caspase 8 and10 may activate caspase 3 directly, or cleave the BH3-containing proteinBid to activate a mitochondria-dependent apoptosis pathway throughrelease of cytochrome C and caspase 9 activation (Desagher, S., and J.C. Martinou. 2000. Trends Cell Biol 10:369-377; Scaffidi, C., et al.1998. Embo J 17:1675-1687). Following the formation of the death domaincomplex, several signal transduction pathways are activated such ascaspase, NF-κB, and JNK/p38. Activation of these signaling pathwaysleads to regulation of death receptor-mediated apoptosis through theBcl-2 and IAP family of proteins.

By “agonist” is meant a substance (molecule, drug, protein, etc.) thatis capable of combining with a receptor (e.g. death receptor) on a celland initiating the same reaction or activity typically produced by thebinding of the endogenous ligand (e.g., apoptosis). The agonist of thepresent method can be a death receptor ligand. Thus, the agonist can beTNF, Fas Ligand, or TRAIL. The agonist can further be a fragment ofthese ligands comprising the death receptor binding domain such that thefragment is capable of binding and activating the death receptor. Theagonist can further be a fusion protein comprising the death receptorbinding domain such that the fusion protein is capable of binding andactivating the death receptor. The agonist can further be a fusionprotein comprising the death receptor binding domain such that thefusion protein is capable of binding and activating the death receptor.The agonist can further be a polypeptide having an amino acid sequencewith at least 85% homology to TNF, Fas or TRAIL such that the homologueis capable of binding and activating the death receptor.

The agonist can further be an apoptosis-inducing antibody that binds thedeath receptor. The “antibody” can be monoclonal, polyclonal, chimeric,single chain, humanized, fully human antibody, or any Fab or F(ab′)2fragments thereof. By “apoptosis-inducing antibody” is meant an antibodythat causes programmed cell death either before or after activationusing the methods provided herein. Thus, the agonist of the presentmethod can be an antibody specific for a Fas, TNFR1 or TRAIL deathreceptor, such that the antibody activates the death receptor. Theagonist can be an antibody specific for DR4 or DR5. The agonist can be aDR5 antibody having the same epitope specificity, or secreted by, amouse-mouse hybridoma having ATCC Accession Number PTA-1428 (e.g., theTRA-8 antibody), ATCC Accession Number PTA-1741 (e.g., the TRA-1antibody), ATCC Accession Number PTA-1742 (e.g., the TRA-10 antibody Theagonist can be an antibody having the same epitope specificity, orsecreted by, the hybridoma having ATCC Accession Number PTA-3798 (e.g.,the 2E12 antibody).

The TRAIL receptor targeted by the antibody of the present method can beDR4 or DR5. Such receptors are described in published patentapplications WO99/03992, WO98/35986, WO98/41629, WO98/32856, WO00/66156,WO98/46642, WO98/5173, WO99/02653, WO99/09165, WO99/11791, WO99/12963and published U.S. Pat. No. 6,313,269, which are all incorporated hereinby reference in their entirety for the receptors taught therein.Monoclonal antibodies specific for these receptors can be generatedusing methods known in the art. See, e.g., Kohler and Milstein, Nature,256:495-497 (1975) and Eur. J. Immunol. 6:511-519 (1976), both of whichare hereby incorporated by reference in their entirety for thesemethods. See also methods taught in published patent applicationWO01/83560, which is incorporated herein by reference in its entirety.

The antibody of the present method can be an antibody known in the art,including, for example, a DR5 antibody having the same epitopespecificity, or secreted by, a mouse-mouse hybridoma having ATCCAccession Number PTA-1428 (e.g., the TRA-8 antibody), ATCC AccessionNumber PTA-1741 (e.g., the TRA-1 antibody), ATCC Accession NumberPTA-1742 (e.g., the TRA-10 antibody). Other examples include an antibodyhaving the same epitope specificity, or secreted by, the hybridomahaving ATCC Accession Number PTA-3798 (e.g., the 2E12 antibody).

By “CARD containing protein” is meant a family of proteins that containa caspase-associated recruitment domain (CARD) and are characterized bythe ability to bind a death receptor, wherein binding is optionallyoutside of the death domain, and modulate the activation of apoptosis bythe death domain of said death receptor. DDX3 is a representative memberof this family. The CARD containing proteins include RNA helicases ofthe DEAD (SEQ ID NO:21) box protein family. The disclosed CARDcontaining protein can be, for example, DDX3 (SEQ ID NO:25, accessionno. gi:13514816), mda-5 (accession no. gi:11344593), or RIG-1 (accessionno. gi:6048564). The CARD containing protein can further be apolypeptide having an amino acid sequence with at least 85% homology toDDX3, mda-5, or RIG-1.

The RNA helicases of the DEAD-box protein family are highly conservedfrom bacteria to mammals, are involved in a variety of metabolicprocesses involving RNA, and are crucial for cell survival (Heinlein, U.A. 1998. J Pathol 184:345-347). All members of this family of proteinshave an ATPase motif that is composed of the characteristic amino acidsequence D-E-A-D (Asp-Glu-Ala-Asp, SEQ ID NO: 21), giving the name tothis family. It is generally believed that DEAD (SEQ ID NO:21) boxproteins are RNA helicases, as ribonucleic acid binding proteins,required for translation initiation, RNA splicing, ribosomal assembly,RNA degradation, mRNA stability and RNA editing. While some of these RNAhelicases play a crucial role in the translation of specialtranscriptional factors, the over-expression of some is related tocarcinogenesis. DDX1 is co-amplified with N-myc in neuroblastomas(George, R. E., et al. 1996. Oncogene 12:1583-1587; Godbout, R., et al.1998. J Biol Chem 273:21161-21168). The RNA helicase, p68, isconsistently overexpressed in tumors as compared with matched normaltissue. The accumulated p68 appears to be poly-ubiquitinated, suggestinga possible defect in proteasome-mediated degradation in these tumors(Causevic, M., et al. 2001. Oncogene 20:7734-7743), suggesting that thedysregulation of p68 expression occurs early during tumor development.The rck/p54 of the DEAD (SEQ ID NO:21) box protein/RNA helicase familymay contribute to cell proliferation and carcinogenesis in thedevelopment of human colorectal tumors at the translational level byincreasing synthesis of c-myc protein (Hashimoto, K., et al. 2001.Carcinogenesis 22:1965-1970). DDX3 is a member of this family althoughthe RNA helicase function of DDX3 is unknown (Fu, J. J., et al. 2002.Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 34:655-661). Ithas been reported that DDX3 could interact with the HCV core protein andregulate the translation of the HCV viral proteins (Owsianka, A. M., andA. H. Patel. 1999. Virology 257:330-3400).

Some of the RNA helicases contain a conserved CARD (caspase recruitmentdomain; Yoneyama, M., et al. 2004. Nat Immunol 5:730-737; Kang, D. C.,et al. 2004. Oncogene 23:1789-1800; Kang, D. C., et al. 2002. Proc NatlAcad Sci USA 99:637-642). Subtraction hybridization identified melanomadifferentiation-associated gene-5 (mda-5) as a gene induced duringdifferentiation, cancer reversion, and programmed cell death(apoptosis). This gene contains both a caspase recruitment domain andputative DExH group RNA helicase domains. Mda-5 may function as amediator of IFN-induced growth inhibition and/or apoptosis (Kang, D. C.,et al. 2002. Proc Natl Acad Sci USA 99:637-642). A more recent studyindicates that the level of mda-5 mRNA is low in normal tissues, whereasexpression is induced in a spectrum of normal and cancer cells byIFN-beta. Expression of mda-5 by means of a replication incompetentadenovirus, Ad.mda-5, induces apoptosis in HO-1 cells as confirmed bymorphologic, biochemical and molecular assays (Kang, D. C., et al. 2004.Oncogene 23:1789-1800). The retinoic acid inducible gene I (RIG-I),which encodes a DExD/H box RNA helicase that contains a caspaserecruitment domain, is an essential regulator of dsRNA-inducedsignaling, as assessed by functional screening and assays. A helicasedomain with intact ATPase activity was responsible for thedsRNA-mediated signaling. The caspase recruitment domain transmitted‘downstream’ signals, resulting in the activation of transcriptionfactors NF-κB and IRF-3. Subsequent gene activation by these factorsinduced antiviral functions, including type I interferon production(Yoneyama, M., et al. 2004. Nat Immunol 5:730-737).

Proteins containing a caspase-associated recruitment domain (CARD) havebeen established as key regulators of cell death. CARD is composed of aconserved alpha-helical bundle found in the N-terminal of pro-domains ofcertain caspases. CARDs can also be found in a variety of otherproteins. Like the death domain proteins, CARDs function as homotypicprotein interaction motifs that allow the communications of proteins viaCARD/CARD interactions. The proteins with a CARD can be eitherpro-apoptotic or anti-apoptotic. The pro-apoptotic CARD proteins includecertain caspases such as caspase 2, 4, and 9, and Apafl, which playimportant roles in the initiation of apoptosis. The representativeanti-apoptotic CARD proteins include cIAP1 and cIAP2, which interactwith the CARD of caspases, and inhibit caspase activation via their BIRdomain. Many aspects of the function of this family of proteins point totheir potential utility as novel drug targets in the treatment ofcancer. Several CARD containing proteins are critical components of theconserved cell death machinery which, when dysregulated, promotesoncogenesis and contributes prominently to tumor resistance tochemotherapy. The pro-apoptotic protein Apafl, which is inactivated insome cancers, is a CARD protein that is indispensable formitochondria-induced apoptosis. Other anti-apoptotic CARD proteins, suchas the proteins of the IAP family, have been shown to protect tumorsfrom cell death stimuli and to be over-expressed in certain forms ofcancer. Therapeutics that activate or inhibit CARD proteins cantherefore be utilized as chemo-sensitizing agents or as modulators ofapoptosis when used in conjunction with conventional chemotherapy.

Resistance to death receptor agonists can be attributed to the activityof the disclosed CARD containing proteins. The present method thereforeprovides a composition that modulates one or more activities of the CARDcontaining protein to prevent said resistance. By “modulates” is meantthe upregulation, downregulation, activation, antagonism, or otherwisealteration in form or function. “Activities” of a protein include, forexample, transcription, translation, intracellular translocation,secretion, phosphorylation by kinases, cleavage by proteases, homophilicand heterophilic binding to other proteins, ubiquitination. As theactivity of the CARD containing protein is due in part tophosphorylation at or near the death receptor binding amino acids, theprovided modulator can be an inhibitor of CARD containing proteinphosphorylation. Thus, the modulator can be an inhibitor of a kinase orphosphatase. As an example, the modulator can be an inhibitor ofglycogen synthase kinase-3 (GSK-3) activity.

GSK-3 is a protein kinase found in a variety of organisms, includingmammals. Two nearly identical forms of GSK-3 exist: GSK-3α and GSK-3β.The inhibitor can be any known or newly discovered GSK-3 inhibitor.Optimally, the GSK-3 inhibitor of the provided method inhibits at leastGSK-3β. The amino acid sequence for human GSK-3β can be accessed atGenbank accession number P49841, and the corresponding nucleotidesequence at accession number NM_(—)002093. For experimental andscreening purposes, it may be desirable to use an animal model. Forexample, the rat GSK-3β sequence may be accessed at Genbank accessionnumber P18266, and the mouse at Genbank accession number AAD39258.

GSK-3 inhibitors, as used herein, are compounds that directly orindirectly reduce the level of GSK-3 activity in a cell, by competitiveor non-competitive enzyme inhibition; by decreasing protein levels, e.g.by a targeted genetic disruption, reducing transcription of the GSK-3gene, increasing protein instability, etc Inhibitors may be smallorganic or inorganic molecules, anti-sense nucleic acids, antibodies orfragments derived therefrom, etc. Other inhibitors of GSK-3 can be foundthrough screening combinatorial or other chemical libraries for theinhibition of GSK-3 activity.

Examples of direct inhibitors of GSK-3 protein include lithium (Li⁺)(Klein et al. 1996), which potently inhibits GSK-3β activity (K_(i)=2mM), but is not a general inhibitor of other protein kinases. Berylliumions (Be²⁺) are stronger inhibitors of GSK-3, inhibiting in themicromolar range. However, this inhibitory effect is not as selective aslithium because it will also inhibit CDK1 at low doses.

GSK-3 inhibitors also include aloisine, aloisine A, kenpaullone.Aloisine (7-n-Butyl-6-(4-methoxyphenyl)[5H]pyrrolo[2,3-b]pyrazine) is apotent, selective, cell-permeable and ATP-competitive inhibitor ofCdkl/B (IC50=700 nM), CdkS/p35 (IC50=1.5 uM) and GSK-3 (IC50=920 nM)(Mettey Y, et al. (2003) J Med. Chem. 46(2):222-36), incorporated hereinby reference in its entirety for teachings related to this molecule.Aloisine A (7-n-Butyl-6-(4-hydroxyphenyl)[5H]pyrrolo[2,3-b]pyrazine) isa cell-permeable compound that acts as a potent, selective, reversible,and ATP-competitive inhibitor of cyclin dependent kinases, c-JunN-terminal kinase (JNK), and glycogen synthase kinase-3 (GSK-3) (GSK-3alpha, IC50=500 nM) (GSK-3 beta, IC50=1.5 uM) (Mettey Y, et al. (2003) JMed. Chem. 46(2):222-36), incorporated herein by reference in itsentirety for teachings related to this molecule. Kenpaullone(9-Bromo-7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-one) is a potent,cell-permeable inhibitor of glycogen synthase kinase-3b (GSK3b, IC50=230nM), Lck and cyclin-dependent kinases (Cdks) (Schultz C, et al. (1999)J. Med. Chem. 42(15):2909-19; Zaharevitz D W, et al (1999) Cancer Res.59(11):2566-9; Kunick C, et al. (2004) J. Med. Chem. 47(1):22-36), whichare all incorporated herein by reference in their entirety for teachingsrelated to this molecule.

A number of other compounds have been found to inhibit GSK-3. Themajority inhibit kinase activity through interaction with theATP-binding site. They include Bisindole- and Anilino maleimides,Aldisine alkaloids, Paullones, Indirubins and Pyraloquinoxalines. Forexample, Paullones and their use in GSK-3 inhibition is described, forexample, in Kunick C, et al. J Med. Chem. 2004 Jan. 1; 47(1):22-36,which is hereby incorporated by reference herein in its entirety for itsteaching of Paullones. Such compounds are effective at nanomolarconcentrations in vitro and low micromolar in vivo. Again, whilst manyhave been shown to be potent, they are not very specific to GSK-3 andcommonly inhibit the related CDKs at similar levels. However, twostructurally distinct maleimides (SB216763 and SB415286) have been shownto be potent and to have high specificity for GSK-3. They caneffectively substitute for lithium as GSK-3 inhibitors in cell studies.Members of the class of compounds termed granulatimides or didemnimideshave also been found to act as GSK-3 inhibitors (International patentapplication WO 99/47522, which is hereby incorporated herein for itsteaching of these compounds).

Some indirect inhibitors of GSK-3 include wortmannin, which activatesprotein kinase B, resulting in the phosphorylation and inhibition ofGSK-3. Isoproterenol, acting primarily through beta3-adrenoreceptors,decreases GSK-3 activity to a similar extent (approximately 50%) asinsulin (Moule et al. 1997). p70 S6 kinase and p90rsk-1 alsophosphorylate GSK-3β, resulting in its inhibition.

GSK-3 can also be selectively targeted using GSK-3-specific peptides.For example, frequently rearranged in advanced T-cell lymphomas 1(FRAT1) is a mammalian homologue of a GSK3-binding protein (GBP).FRATtide (a peptide corresponding to residues 188-226 of FRAT1) binds toGSK3 and blocks the GSK3-catalysed phosphorylation of Axin andbeta-catenin (Thomas G M, et al. FEBS Lett. 1999 Sep. 17;458(2):247-51).

The GSK-3 inhibitor of the provided method can also be a functionalnucleic acid. Functional nucleic acids are nucleic acid molecules thathave a specific function, such as binding a target molecule orcatalyzing a specific reaction. Functional nucleic acid molecules can bedivided into the following categories, which are not meant to belimiting. For example, functional nucleic acids include antisensemolecules, aptamers, ribozymes, triplex forming molecules, RNAi, andexternal guide sequences. The functional nucleic acid molecules can actas affectors, inhibitors, modulators, and stimulators of a specificactivity possessed by a target molecule, or the functional nucleic acidmolecules can possess a de novo activity independent of any othermolecules.

As CARD containing proteins can be cleaved during death receptor-inducedapoptosis, the modulator of the present method can act by promotingcleavage of the CARD containing protein. As an example, DDX3 isdisassociated from DR5 and cleaved during TRA-8-induced apoptosis (FIGS.9A and C) in parallel with the recruitment of FADD (FIG. 9E). One of thecleavage sites for DDX3 is a relatively conserved DEDD (SEQ ID NO:7)motif between amino acid residues 132-135, which can be cleaved bycaspases 2, 3, 7 or 10. Thus, the modulator can be a caspase or aderivative of a caspase that cleaves a CARD containing protein (e.g.DDX3).

As the activity of the CARD containing protein is dependent upon itsbinding to the death receptor, the modulator of the present method canbe an inhibitor of the interaction between the CARD containing proteinand the death receptor. In one instance, the modulator is a substance(drug, molecule, polypeptide, etc.) that binds a CARD containing proteinat the death receptor-binding site. Thus, the modulator can be apolypeptide comprising the amino acids of the death receptorcorresponding to the binding site of the CARD containing protein. Forexample, the modulator can be a polypeptide comprising amino acids250-340 of DR5 (SEQ ID NO:22, accession no. gi:3721878). Thus, themodulator can comprise the amino acid sequence SEQ ID NO:23. Themodulator can further be a polypeptide comprising a fragment of aminoacid sequence SEQ ID NO:23, such that the fragment is capable of bindingDDX3. As an example, the modulator can be a polypeptide comprising aminoacids 280-310 of DR5 (SEQ ID NO:22, accession no. gi:3721878). Thus, themodulator can comprise the amino acid sequence SEQ ID NO:24. As anotherexample, the modulator can be a polypeptide comprising amino acids300-330 of DR5 (SEQ ID NO:22, accession no. gi:3721878). Thus, themodulator can comprise the amino acid sequence SEQ ID NO:36.

Alternatively, the modulator can be a substance (drug, molecule,polypeptide, etc.) that binds the CARD containing protein binding siteof the death receptor without inhibiting apoptosis. The modulator can bea polypeptide comprising the amino acids corresponding to the deathreceptor-binding site of the CARD containing protein.

The modulator of the present method can affect the ability of a CARDcontaining protein to prevent the activation of capase-dependentapoptosis. The CARD domain of CARD containing proteins is involved inthe recruitment of inhibitors of apoptosis (IAP), which suppressapoptosis in host cells during viral infection (Crook, N. E., et al.1993. J Virol 67:2168-2174). The IAP family antagonizes cell death byinteracting with and inhibiting the enzymatic activity of maturecaspases. Eight distinct mammalian IAPs have been identified, includingXIAP, c-IAP1, c-IAP2, and ML-IAP/Livin (see, for example, Ashhab, Y., etal. 2001. FEBS Lett 495:56-60; Kasof, G. M., and B. C. Gomes. 2001. JBiol Chem 276:3238-3246; Vucic, D., et al. 2000. Curr Biol 10:1359-1366,which are all incorporated herein by references in their entirety asrelated to these IAP molecules). All IAPs contain one to threebaculovirus IAP repeat (BIR) domains and have homologous sequence(CX2CX16HX6C). Through the BIR domain, IAP molecules bind and directlyinhibit caspases (Deveraux, Q. L., and J. C. Reed. 1999. Genes Dev13:239-252; Deveraux, Q. L., et al. 1997. Nature 388:300-304; Deveraux,Q. L., and J. C. Reed. 1999. Genes Dev 13:239-252, which are allincorporated herein by references in their entirety as related to theinteraction of IAPs and caspases). The mitochondrial proteinsSmac/DIABLO could bind to and antagonize IAPs (Suzuki, Y., et al. 2001.J Biol Chem 276:27058-27063) to suppress IAP function (Wieland, I., etal. 2000. Oncol Res 12:491-500) (The cited references are allincorporated herein by references in their entirety as related to theinhibition of IAPs). Thus, the modulator of the present method can be aninhibitor of CARD-dependent binding. The modulator can affect theability of a CARD containing protein to recruit a caspase or modulatorof caspase such as IAP. The modulator can be a substance (drug,molecule, polypeptide, fusion protein, antibody, antibody fragment,etc.) that binds a CARD containing protein such that the CARD containingprotein has reduced binding and recruitment of IAPs. The modulator canbe a CARD containing protein-binding fragment of, for example,caspase-1, caspase-2, caspase-4 or caspase-5, cIAP1, cIAP2, XIAP, orsurvivin.

The modulator can further be an inhibitor of IAP or CARD containingprotein gene expression in the target cell. There are various knownmethods of inhibiting the expression of a protein in a cell, includingtriplex forming molecules, ribozymes, external guide sequences,antisense molecules, and RNAi molecules.

Antisense molecules are designed to interact with a target nucleic acidmolecule through either canonical or non-canonical base pairing. Theinteraction of the antisense molecule and the target molecule isdesigned to promote the destruction of the target molecule through, forexample, RNAseH mediated RNA-DNA hybrid degradation. Alternatively theantisense molecule is designed to interrupt a processing function thatnormally would take place on the target molecule, such as transcriptionor replication. Antisense molecules can be designed based on thesequence of the target molecule. Numerous methods for optimization ofantisense efficiency by finding the most accessible regions of thetarget molecule exist. Exemplary methods would be in vitro selectionexperiments and DNA modification studies using DMS and DEPC. It ispreferred that antisense molecules bind the target molecule with adissociation constant (K_(d)) less than or equal to 10-6, 10-8, 10-10,or 10-12. A representative sample of methods and techniques which aid inthe design and use of antisense molecules can be found in U.S. Pat. Nos.5,135,917, 5,294,533, 5,627,158, 5,641,754, 5,691,317, 5,780,607,5,786,138, 5,849,903, 5,856,103, 5,919,772, 5,955,590, 5,990,088,5,994,320, 5,998,602, 6,005,095, 6,007,995, 6,013,522, 6,017,898,6,018,042, 6,025,198, 6,033,910, 6,040,296, 6,046,004, 6,046,319, and6,057,437, which are herein incorporated by reference in their entiretyfor methods and techniques regarding antisense molecules.

Aptamers are molecules that interact with a target molecule, preferablyin a specific way. Typically aptamers are small nucleic acids rangingfrom 15-50 bases in length that fold into defined secondary and tertiarystructures, such as stem-loops or G-quartets. Aptamers can bind smallmolecules, such as ATP (U.S. Pat. No. 5,631,146) and theophiline (U.S.Pat. No. 5,580,737), as well as large molecules, such as reversetranscriptase (U.S. Pat. No. 5,786,462) and thrombin (U.S. Pat. No.5,543,293). Aptamers can bind very tightly with K_(d)s from the targetmolecule of less than 10⁻¹² M. It is preferred that the aptamers bindthe target molecule with a K_(d) less than 10⁻⁶, 10⁻⁸, 10⁻¹⁰, or 10⁻¹².Aptamers can bind the target molecule with a very high degree ofspecificity. For example, aptamers have been isolated that have greaterthan a 10,000 fold difference in binding affinities between the targetmolecule and another molecule that differ at only a single position onthe molecule (U.S. Pat. No. 5,543,293). It is preferred that the aptamerhave a K_(d) with the target molecule at least 10, 100, 1000, 10,000, or100,000 fold lower than the K_(d) with a background binding molecule. Itis preferred when doing the comparison for a polypeptide for example,that the background molecule be a different polypeptide. Representativeexamples of how to make and use aptamers to bind a variety of differenttarget molecules can be found in U.S. Pat. Nos. 5,476,766, 5,503,978,5,631,146, 5,731,424, 5,780,228, 5,792,613, 5,795,721, 5,846,713,5,858,660, 5,861,254, 5,864,026, 5,869,641, 5,958,691, 6,001,988,6,011,020, 6,013,443, 6,020,130, 6,028,186, 6,030,776, and 6,051,698,which are herein incorporated by reference in their entirety for methodsand techniques regarding aptamers.

Ribozymes are nucleic acid molecules that are capable of catalyzing achemical reaction, either intramolecularly or intermolecularly.Ribozymes are thus catalytic nucleic acid. It is preferred that theribozymes catalyze intermolecular reactions. There are a number ofdifferent types of ribozymes that catalyze nuclease or nucleic acidpolymerase type reactions which are based on ribozymes found in naturalsystems, such as hammerhead ribozymes, (U.S. Pat. Nos. 5,334,711,5,436,330, 5,616,466, 5,633,133, 5,646,020, 5,652,094, 5,712,384,5,770,715, 5,856,463, 5,861,288, 5,891,683, 5,891,684, 5,985,621,5,989,908, 5,998,193, 5,998,203; International Patent Application Nos.WO 9858058 by Ludwig and Sproat, WO 9858057 by Ludwig and Sproat, and WO9718312 by Ludwig and Sproat) hairpin ribozymes (for example, U.S. Pat.Nos. 5,631,115, 5,646,031, 5,683,902, 5,712,384, 5,856,188, 5,866,701,5,869,339, and 6,022,962), and tetrahymena ribozymes (for example, U.S.Pat. Nos. 5,595,873 and 5,652,107). There are also a number of ribozymesthat are not found in natural systems, but which have been engineered tocatalyze specific reactions de novo (for example, U.S. Pat. Nos.5,580,967, 5,688,670, 5,807,718, and 5,910,408). Preferred ribozymescleave RNA or DNA substrates, and more preferably cleave RNA substrates.Ribozymes typically cleave nucleic acid substrates through recognitionand binding of the target substrate with subsequent cleavage. Thisrecognition is often based mostly on canonical or non-canonical basepair interactions. This property makes ribozymes particularly goodcandidates for target specific cleavage of nucleic acids becauserecognition of the target substrate is based on the target substratessequence. Representative examples of how to make and use ribozymes tocatalyze a variety of different reactions can be found in U.S. Pat. Nos.5,646,042, 5,693,535, 5,731,295, 5,811,300, 5,837,855, 5,869,253,5,877,021, 5,877,022, 5,972,699, 5,972,704, 5,989,906, and 6,017,756.

Triplex forming functional nucleic acid molecules are molecules that caninteract with either double-stranded or single-stranded nucleic acid.When triplex molecules interact with a target region, a structure calleda triplex is formed, in which there are three strands of DNA forming acomplex dependant on both Watson-Crick and Hoogsteen base-pairing.Triplex molecules are preferred because they can bind target regionswith high affinity and specificity. It is preferred that the triplexforming molecules bind the target molecule with a K_(d) less than 10-6,10-8, 10-10, or 10-12. Representative examples of how to make and usetriplex forming molecules to bind a variety of different targetmolecules can be found in U.S. Pat. Nos. 5,176,996, 5,645,985,5,650,316, 5,683,874, 5,693,773, 5,834,185, 5,869,246, 5,874,566, and5,962,426.

External guide sequences (EGSs) are molecules that bind a target nucleicacid molecule forming a complex, and this complex is recognized by RNaseP, which cleaves the target molecule. EGSs can be designed tospecifically target a RNA molecule of choice. RNAse P aids in processingtransfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited tocleave virtually any RNA sequence by using an EGS that causes the targetRNA:EGS complex to mimic the natural tRNA substrate. (WO 92/03566 byYale, and Forster and Altman, Science 238:407-409 (1990)).

Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA can beutilized to cleave desired targets within eukarotic cells. (Yuan et al.,Proc. Natl. Acad. Sci. USA 89:8006-8010 (1992); WO 93/22434 by Yale; WO95/24489 by Yale; Yuan and Altman, EMBO J. 14:159-168 (1995), andCarrara et al., Proc. Natl. Acad. Sci. (USA) 92:2627-2631 (1995)).Representative examples of how to make and use EGS molecules tofacilitate cleavage of a variety of different target molecules be foundin U.S. Pat. Nos. 5,168,053, 5,624,824, 5,683,873, 5,728,521, 5,869,248,and 5,877,162.

Gene expression can also be effectively silenced in a highly specificmanner through RNA interference (RNAi). This silencing was originallyobserved with the addition of double stranded RNA (dsRNA) (Fire, A., etal. (1998) Nature, 391:806-11; Napoli, C., et al. (1990) Plant Cell2:279-89; Hannon, G. J. (2002) Nature, 418:244-51). Once dsRNA enters acell, it is cleaved by an RNase III—like enzyme, Dicer, into doublestranded small interfering RNAs (siRNA) 21-23 nucleotides in length thatcontains 2 nucleotide overhangs on the 3′ ends (Elbashir, S. M., et al.(2001) Genes Dev., 15:188-200; Bernstein, E., et al. (2001) Nature,409:363-6; Hammond, S. M., et al. (2000) Nature, 404:293-6). In an ATPdependent step, the siRNAs become integrated into a multi-subunitprotein complex, commonly known as the RNAi induced silencing complex(RISC), which guides the siRNAs to the target RNA sequence (Nykanen, A.,et al. (2001) Cell, 107:309-21). At some point the siRNA duplex unwinds,and it appears that the antisense strand remains bound to RISC anddirects degradation of the complementary mRNA sequence by a combinationof endo and exonucleases (Martinez, J., et al. (2002) Cell, 110:563-74).However, the effect of iRNA or siRNA or their use is not limited to anytype of mechanism.

Short Interfering RNA (siRNA) is a double-stranded RNA that can inducesequence-specific post-transcriptional gene silencing, therebydecreasing or even inhibiting gene expression. In one example, an siRNAtriggers the specific degradation of homologous RNA molecules, such asmRNAs, within the region of sequence identity between both the siRNA andthe target RNA. For example, WO 02/44321 discloses siRNAs capable ofsequence-specific degradation of target mRNAs when base-paired with 3′overhanging ends, herein incorporated by reference for the method ofmaking these siRNAs. Sequence specific gene silencing can be achieved inmammalian cells using synthetic, short double-stranded RNAs that mimicthe siRNAs produced by the enzyme dicer (Elbashir, S. M., et al. (2001)Nature, 411:494 498) (Ui-Tei, K., et al. (2000) FEBS Lett 479:79-82).siRNA can be chemically or in vitro-synthesized or can be the result ofshort double-stranded hairpin-like RNAs (shRNAs) that are processed intosiRNAs inside the cell. Synthetic siRNAs are generally designed usingalgorithms and a conventional DNA/RNA synthesizer. Suppliers includeAmbion (Austin, Tex.), ChemGenes (Ashland, Mass.), Dharmacon (Lafayette,Colo.), Glen Research (Sterling, Va.), MWB Biotech (Esbersberg,Germany), Proligo (Boulder, Colo.), and Qiagen (Vento, The Netherlands).siRNA can also be synthesized in vitro using kits such as Ambion'sSILENCER® siRNA Construction Kit. Disclosed herein are any siRNAdesigned as described above based on the sequences for c-Kit or SCF. Forexample, siRNAs for silencing gene expression of c-Kit is commerciallyavailable (SURESILENCING™ Human c-Kit siRNA; Zymed Laboratories, SanFrancisco, Calif.).

The production of siRNA from a vector is more commonly done through thetranscription of a short hairpin RNAs (shRNAs). Kits for the productionof vectors comprising shRNA are available, such as, for example,Imgenex's GENESUPPRESSOR™ Construction Kits and Invitrogen's BLOCK-IT™inducible RNAi plasmid and lentivirus vectors. Disclosed herein are anyshRNA designed as described above based on the sequences for the hereindisclosed inflammatory mediators.

Thus, the modulator of the present method can comprise siRNA or shRNA.The modulator can be an inhibitor of cIAP1 (accession no. gi:41349435,cIAP2 (accession no. gi:33946283, XIAP (accession no gi:1184319),survivin (accession no gi:2315862), DDX3 (SEQ ID NO:25, accession no.gi:13514816), mda-5 (accession no. gi:11344593), or RIG-1 (accession no.gi:6048564) gene expression. Thus, the modulator can comprise a shRNAderived from the nucleic acid sequence of DDX3 (SEQ ID NO:25, accessionno. gi:13514816). As an example, the modulator can comprise a shRNAencoded by the nucleic acid sequence SEQ ID NO:10, 12, 14, or 16.

There are several transfection reagents that can be used for thedelivery of siRNA to a cell, such as, for example, Invitrogen'sLipofectamine 2000, Mirus' TranslT-TKO, and Novagen's RiboJuice siRNATransfection Reagent. However, transfection reagents generally do notwork in vivo. Naked siRNA can be delivered directly into the vasculatureof a subject, which has the advantage that no other proteins aredelivered or expressed, which is critical as nucleic acids are notimmunogenic. There are also methods of delivering nucleic acids acrossepithelial barriers such as the skin using some form of energy todisrupt the epithelium, e.g. sonophoresis (U.S. Pat. No. 5,421,816, U.S.Pat. No. 5,618,275, U.S. Pat. No. 6,712,805 and U.S. Pat. No. 6,487,447,which are all incorporated herein by reference for their teaching ofultrasound mediated delivery of compounds through the skin).

Provided is a method of screening a cell for a biomarker of resistanceto a death receptor agonist comprising assaying the cell for total DDX3,or a homologue thereof. Also, provided is a method of screening a cellfor a biomarker of resistance to a death receptor agonist comprisingassaying the association of the death receptor and a CARD containingprotein, wherein high levels of association signify resistance to theagonist. Association between the death receptor and CARD containingprotein indicates resistance to the agonist. Optionally, the cell to bescreened is pre-contacted with a death receptor agonist (e.g. agonisticantibody). Thus, provided is a method of screening a cell for abiomarker of resistance to a death receptor agonist comprisingcontacting the cell with the death receptor agonist and monitoring thefractional association of the death receptor and a CARD containingprotein, wherein association signifies resistance to the agonist.Optionally, in the various methods involving detecting association, onecould measure dissociation and substract the dissociated amount from thetotal to calculate the associated amount.

The contacting step of the present method can be done either in vivo orin vitro. Monitoring of the association between the death receptor andCARD containing protein can involve the isolation of a protein fragment(e.g. death receptor) from the cell(s) using specific antibodies (e.g.by immunoprecipitation). This method can further involve the analysis ofthe protein fragment for associated proteins (e.g. DDX3) by standardimmunodetection methods, such as Western blot, radioimmunoassay (RIA),or ELISA. These antibody-based methods are well known in the art and areeasily tailored to each death receptor, CARD containing protein, caspaseand modulator of caspase of interest. As an illustration, the presentmethod can comprise treating a cell from a subject with TRA-8 antibody,isolating the protein from the cell lysate, immunoprecipitating DR5protein, separating DR5 by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) (reducing or non-reducing conditions), transferring theseparated protein to a nitrocellulose membrane, and using standardWestern blot techniques to detect DDX3 associated with DR5, wherein theassociation is evidence of TRA-8 resistance in that cell.

Also provided is a method of screening a cell for a biomarker ofresistance to a death receptor agonist comprising monitoring theassociation of a caspase or modulator of caspases (eg, cIAP1, cIAP2,XIAP, survivin) with the CARD containing protein and comparing the levelof association with a sample from known resistant and non-resistantcontrol cells. The association of IAPs with the CARD containing proteinat levels similar to that of resistant cells signifies resistance to theagonist. Optionally, the cell to be screened is pre-contacted with adeath receptor agonist (e.g. agonistic antibody).

As an illustration, the present method can comprise treating a cell froma subject with TRA-8 antibody, isolating the protein from the celllysate, immunoprecipitating DDX3 protein, separating DDX3 bySDS-polyacrylamide gel electrophoresis (SDS-PAGE) (reducing ornon-reducing conditions), transferring the separated protein to anitrocellulose membrane, and using standard Western techniques to detectcaspases (e.g. caspase-1, caspase-2, caspase-4, caspase-5) and IAPs(e.g. cIAP1, cIAP2, XIAP, survivin) associated with DDX3, wherein thedetection of IAPs with DDX3 is evidence of TRA-8 resistance in thatcell. The level of association can be compared to a control level. Thecontrol level can be based on non-resistant cells. If the test level ishigher than that of non-resistant control cells, then resistance isindicated. The control level can be based on resistant cells such that asimilarity between the test levels and control levels indicatesresistance.

Also provided is a method of screening for a modulator of a CARDcontaining protein. In particular, provided herein is such a screeningmethod wherein the modulator reverses or prevents a target cell'sresistance to a death receptor agonist. The steps of the screeningmethod comprise contacting the CARD containing protein with a candidateagent and detecting a change (e.g. decrease) in one or more activitiesof the CARD containing protein in the presence of the candidate agent ascompared to the absence of the candidate agent, wherein the activity oractivities correlate with the target cell's resistance to the deathreceptor agonist. A decrease in the activity or activities of the CARDcontaining protein indicates the candidate agent modulates the CARDcontaining protein. This method could be modified to utilize a modifiedCARD containing protein, including for example, naturally occurringmodifications or non-naturally occurring modifications. Suchmodifications can include truncations, mutations, chimeric proteins,etc. For example, the nucleic acid sequence for DDX3 is set forth in SEQID NO:25. Examples of DDX3 mutations include adenosine to guanosinesubstitutions at positions 1842 and 2493 in SEQ ID NO:25.

Any number of activities of the CARD containing protein can be assessedin the screening methods described herein. For example, the activity ofthe CARD containing protein can be phosphorylation, including forexample, phosphorylation at or near the death receptor binding aminoacids. Thus, the present method can comprise detecting phosphorylationof the CARD containing protein. Cell-based and cell-free assays fordetecting phosphorylation of proteins are well known in the art andinclude the use of antibodies, including, for example,anti-Phosphoserine (Chemicon® AB1603) (Chemicon, Temecula, Calif.),anti-Phosphothreonine (Chemicon® AB1607), and anti-Phosphotyrosine(Chemicon® AB1599). Site-specific antibodies can also be generatedspecific for the phosphorylated form of DDX-3. The methods of generatingand using said antibodies are well known in the art.

Another CARD containing protein activity that can be assessed in thescreening methods described herein is binding activity. For example, theactivity of the CARD containing protein can be binding to the deathreceptor. Thus, the present method can comprise detecting theinteraction between the CARD containing protein and the death receptor.The activity of the CARD containing protein can be CARD-dependentbinding. Thus, the present method can comprise detecting CARD-dependentbinding to, for example, cIAP1, cIAP2, XIAP, or survivin. Methods forthe detection of protein binding are well known in the art and include,for example, co-immunoprecipitation combined with enzyme linkedimmunosorbent assays (ELISAs) or Western blotting. In another example, asandwich assay can be used wherein a first antibody captures the deathreceptor and wherein a second antibody detects the CARD containingprotein. In another example, a sandwich assay can be used wherein afirst antibody captures the CARD containing protein and wherein a secondantibody detects the death receptor.

Further provided herein are the screening assays wherein the assessedactivity of the CARD containing protein is cleavage, including forexample, cleavage that occurs during death receptor-induced apoptosis.Thus, the screening method can comprise detecting cleavage of the CARDcontaining protein. Methods for the detection of protein cleavage arewell known in the art and include, for example, Western blotting.

The contacting step of the screening method can be done either in vivoor in vitro. The screening method can be either cell-based or cell-free.Thus, in one aspect, the CARD containing protein is in a target cell.The CARD containing protein can be naturally occurring in the cell orthe cell can be genetically engineered to produce the CARD containingprotein. In a cell free method, the CARD containing protein can bemodified to be attached to a substrate or to form a chimeric protein.

Optionally, the screening method can further comprise contacting thetarget cell, or a non-cellular system comprising a death receptor, oneor more times with a death receptor agonist and detecting the level ofresistance to the death receptor agonist. The level of resistance to thedeath receptor agonist can be detected, for example, by measuringapoptosis, a decline in apoptosis upon repeated exposure to the deathreceptor agonist indicating an increase in resistance. Methods for thedetection of apoptosis are well known in the art and include, forexample, reagents for detecting terminal dUTP nick-end labeling (TUNEL),active-caspase 3, cell surface phospholipid phosphatidylserine (PS) byAnnexin V. Reagents for these and other methods for detecting apoptosisare commercially available.

In general, candidate agents can be identified from large libraries ofnatural products or synthetic (or semi-synthetic) extracts or chemicallibraries according to methods known in the art. Those skilled in thefield of drug discovery and development will understand that the precisesource of test extracts or compounds is not critical to the screeningprocedure(s) of the invention. Accordingly, virtually any number ofpeptides, chemical extracts or compounds can be screened using theexemplary methods described herein. Examples of such peptides, extractsor compounds include, but are not limited to, plant-, fungal-,prokaryotic- or animal-based extracts, fermentation broths, andsynthetic compounds, as well as modification of existing compounds.Numerous methods are also available for generating random or directedsynthesis (e.g., semi-synthesis or total synthesis) of any number ofchemical compounds, including, but not limited to, saccharide-, lipid-,peptide-, polypeptide- and nucleic acid-based compounds. Syntheticcompound libraries are commercially available, e.g., from BrandonAssociates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.).Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant, and animal extracts are commercially available from anumber of sources, including Biotics (Sussex, UK), Xenova (Slough, UK),Harbor Branch Oceangraphics Institute (Ft. Pierce, Fla.), and PharmaMar,U.S.A. (Cambridge, Mass.). In addition, natural and syntheticallyproduced libraries are produced, if desired, according to methods knownin the art, e.g., by standard extraction and fractionation methods.Furthermore, if desired, any library or compound is readily modifiedusing standard chemical, physical, or biochemical methods. In addition,those skilled in the art of drug discovery and development readilyunderstand that methods for dereplication (e.g., taxonomicdereplication, biological dereplication, and chemical dereplication, orany combination thereof) or the elimination of replicates or repeats ofmaterials already known for their effect on an activity of the CARDcontaining protein should be employed whenever possible.

When a crude extract is found to have a desired activity, furtherfractionation of the positive lead extract is necessary to isolatechemical constituents responsible for the observed effect. Thus, thegoal of the extraction, fractionation, and purification process is thecareful characterization and identification of a chemical entity withinthe crude extract having an activity that stimulates or inhibits anactivity of the CARD containing protein. The same assays describedherein for the detection of activities in mixtures of compounds can beused to purify the active component and to test derivatives thereof.Methods of fractionation and purification of such heterogenous extractsare known in the art. If desired, compounds shown to be useful agentsfor treatment are chemically modified according to methods known in theart. Compounds identified as being of therapeutic value may besubsequently analyzed using animal models for diseases or conditions inwhich it is desirable to regulate or mimic an activity of the CARDcontaining protein.

Provided is a method of monitoring resistance to a death receptoragonist in a subject, comprising acquiring a biological sample from thesubject and detecting association of a CARD containing protein with adeath receptor in the sample, the association indicating resistance. Asdescribed above, the level of association can be compared to a controllevel.

As an illustration, the present method can comprise isolating from asubject a biological sample, wherein the subject has been treated with atherapeutic anti-DR5 antibody (e.g. TRA-8), isolating the protein fromthe biological sample, immunoprecipitating DR5 protein, separating DR5by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (reducing ornon-reducing conditions), transferring the separated protein to anitrocellulose membrane, and using antibodies to DDX3 and standardWestern blot techniques to detect DDX3 associated with DR5, whereinassociation is evidence of TRA-8 resistance in that cell.

Provided is a method of monitoring resistance to a death receptoragonist in a subject, comprising acquiring a biological sample from thesubject and detecting association of a caspase or modulator of caspasewith a CARD containing protein in the sample, the association indicatingresistance.

As an illustration, the present method can comprise isolating from asubject a biological sample being treated with a therapeutic anti-DR5antibody (e.g. TRA-8), isolating the protein from the biological sample,immunoprecipitating DDX-3 protein, separating DDX-3 bySDS-polyacrylamide gel electrophoresis (SDS-PAGE) (reducing ornon-reducing conditions), transferring the separated protein to anitrocellulose membrane, and using standard Western techniques to detectcaspases (e.g., caspase-1, caspase-2, caspase-4, caspase-5) and IAPs(e.g., cIAP1, cIAP2, XIAP, survivin) associated with DDX3, wherein thedetection of cIAP1 with DDX3, for example, is evidence of TRA-8resistance in that cell.

Provided is a method of monitoring resistance to a death receptoragonist in a subject, comprising acquiring a biological sample from thesubject and detecting phosphorylation of DDX3. Methods for detectingphosphorylation of proteins are well known in the art and include theuse of antibodies, including, for example, anti-Phosphoserine (Chemicon®AB1603), anti-Phosphothreonine (Chemicon® AB1607), andanti-Phosphotyrosine (Chemicon® AB1599). Site-specific antibodies canalso be generated specific for the phosphorylated form of DDX-3. Themethods of generating and using said antibodies are well known in theart.

Provided is a method of selectively inducing apoptosis in a target cellexpressing a death receptor, comprising the steps of contacting thetarget cell with a therapeutic amount of a death receptor agonist thatspecifically binds the death receptor and administering to the targetcell a therapeutic amount of a modulator of one or more activities of aCARD containing protein.

The ability of an agonist and CARD containing protein modulator toinduce apoptosis can be confirmed by culturing cells such as the humanleukemia cell line Jurkat (American Type Culture No. TIB-152) andastrocytoma cell line 1321N1 in medium in which the test sample has beenadded. The survival rate can be determined by, for example, using anATPLITE assay.

The methods and compositions provided herein can be used in thetreatment of diseases associated with inappropriate survival orproliferation of cells, including those attributable to dysregulation ofthe apoptosis systems in cancer or in inflammatory and autoimmunediseases. Inflammatory and autoimmune diseases illustratively includesystemic lupus erythematosus, Hashimoto's disease, rheumatoid arthritis,graft-versus-host disease, Sjögren's syndrome, pernicious anemia,Addison disease, scleroderma, Goodpasture's syndrome, Crohn's disease,autoimmune hemolytic anemia, sterility, myasthenia gravis, multiplesclerosis, Basedow's disease, thrombopenia purpura, insulin-dependentdiabetes mellitus, allergy, asthma, atopic disease, arteriosclerosis,myocarditis, cardiomyopathy, glomerular nephritis, hypoplastic anemia,rejection after organ transplantation. Cancers include numerousmalignancies of lung, prostate, liver, ovary, colon, cervix, lymphaticand breast tissues. Thus, the provided compositions and methods canfurther be used to target and selectively induce apoptosis in activatedimmune cells including activated lymphocytes, lymphoid cells, myeloidcells, and rheumatoid synovial cells (including inflammatorysynoviocytes, macrophage-like synoviocytes, fibroblast-likesynoviocytes) and in virally infected cells (including those infectedwith HIV, for example) so long as those targeted cells express or can bemade to express the specific death receptors (i.e., DR4 or DR5).

Provided is a method of treating a subject with cancer or with anautoimmune or inflammatory diesase, comprising administering to thesubject a therapeutic amount of a death receptor agonist and a modulatorof one or more activities of a CARD containing protein, wherein themodulator reduces resistance to the death receptor agonist.

As used throughout, a “therapeutic amount” of a death receptor agonistand/or modulator of CARD containing protein is the quantity sufficientto cause apoptosis in the target cell. As used herein, the terms“therapeutic amount” and “pharmaceutically effective amount” aresynonymous. One of skill in the art could readily determine the propertherapeutic amount.

In the treatment of disease, e.g., cancer, autoimmune and inflammatorydiseases, combinations of treatment can also be used. For example, theagonists and modulators of CARD containing protein of the providedmethods and compositions can be administered in conjunction with othertherapeutic agents. As used herein a “therapeutic agent” is a compoundor composition effective in ameliorating a pathological condition.Radiotherapy can also be combined with or without other therapeuticagents. One skilled in the art would adapt the form of radiotherapy tothe disease.

Examples of therapeutic agents include chemotherapeutic agents,anti-inflammatory agents, Disease Modifying Anti Rheumatic Drug(DMARDs), antibodies, members of TNF family, antiviral agents,anti-opportunistic agents, antibiotics, immunosuppresives,immunoglobulins, anti-malarial agents, anti-rheumatoid arthritis agents,cytokines, chemokines, growth factors, and anti-cancer compounds. Ananti-cancer compound is a compound or composition effective ininhibiting or arresting the growth of an abnormally growing cell.Illustrative examples of anti-cancer compounds include: bleomycin,carboplatin, chlorambucil, cisplatin, colchicine, cyclophosphamide,daunorubicin, dactinomycin, diethylstilbestrol doxorubicin, etoposide,5-fluorouracil, floxuridine, melphalan, methotrexate, mitomycin,6-mercaptopurine, teniposide, 6-thioguanine, vincristine andvinblastine. Further examples of anti-cancer compounds and therapeuticagents are found in The Merck Manual of Diagnosis and Therapy, 15th Ed.,Berkow et al., eds., 1987, Rahway, N.J. and Sladek et al. Metabolism andAction of Anti-Cancer Drugs, 1987, Powis et al. eds., Taylor andFrancis, New York, N.Y.

The PKC inhibitor, bisindolymaleimide VIII (BisVIII), greatlyfacilitates Fas-mediated apoptosis (Zhou, T., et al. 1999. Nat Med5:42-48). It has been shown that that synergistic activation of theJNK/p38 pathway plays an important role (Ohtsuka, T., and T. Zhou. 2002.J Biol Chem 277:29294-29303), and that the enhancement of DR5-mediatedapoptosis by three common chemotherapeutic agents appears to occurthrough a similar mechanism (Ohtsuka, T., D. et al. 2003. Oncogene22:2034-2044). Thus, the provided methods can further comprise the useof apoptosis-inducing compounds, such as bisindolylmaleimide VIII(BisVIII) or other sensitizing agents like SN-50 or LY294002. Thus, theagonists and modulators of CARD containing protein of the providedmethods and compositions can be combined with BisVIII. The agonists andmodulators of CARD containing protein of the provided methods andcompositions can further be combined with a non-steroidalanti-inflammatory drug (NSAID) (e.g., sulindac sulfide or other COX-1 orCOX-2 inhibitors).

Therapy using the agonists of the provided methods and compositions canalso be combined with therapy using other agonists. For example, anantibody to DR5 can be administered to a subject in need thereof alongwith, prior to, or following administration of an antibody to DR4. Suchcombined antibody therapy can be further combined with administration ofone or more of the modulators of CARD containing protein provided hereinand can be further combined with other therapeutic agents.

Provided is a composition comprising a death receptor agonist and anagent that modulates one or more activities of a CARD containingprotein, wherein the modulator reduces resistance to the death receptoragonist. The provided composition can further comprise a therapeuticagent selected from the group consisting of a chemotherapeutic agent,member of TNF family, antiviral agent, anti-inflammatory agent,anti-opportunistic agent, antibiotic, immunosuppresant, immunoglobulin,anti-malarial agent, anti-rheumatoid arthritis agent, cytokine,chemokine, and growth factor.

The term “protein,” “peptide,” “polypeptide,” or “peptide portion” areused interchangeably herein and are used broadly herein to mean two ormore amino acids linked by a peptide bond. The term “fragment” is usedherein to refer to a portion of a full-length polypeptide or protein,such portion which can be produced by a proteolytic reaction on apolypeptide, i.e., a peptide produced upon cleavage of a peptide bond inthe polypeptide. It should be recognized that the fragment need notnecessarily be produced by a proteolytic reaction but can be producedusing methods of chemical synthesis or methods of recombinant DNAtechnology, to produce a synthetic polypeptide. It should be recognizedthat the term “protein” and “polypeptide” are not used herein to suggesta particular size or number of amino acids comprising the molecule andthat a peptide of the invention can contain up to several amino acidresidues or more.

By “isolated” or “purified” is meant a composition (e.g., a polypeptideor nucleic acid) that is substantially free from other materials,inlcuding materials with which the composition is normally associated innature. The polypeptides of the invention, or fragments thereof, can beobtained, for example, by extraction from a natural source (e.g.,phage), by expression of a recombinant nucleic acid encoding thepolypeptide (e.g., in a cell or in a cell-free translation system), orby chemically synthesizing the polypeptide. In addition, polypeptidefragments may be obtained by any of these methods, or by cleaving fulllength polypeptides. A fragment of a reference protein or polypeptideincludes only contiguous amino acids of the referenceprotein/polypeptide, and is at least one amino acid shorter than thereference sequence.

When specific proteins are referred to herein, variants, derivatives,and fragements are contemplated. Protein variants and derivatives arewell understood to those of skill in the art and in can involve aminoacid sequence modifications. For example, amino acid sequencemodifications typically fall into one or more of three classes:substitutional, insertional or deletional variants. Insertions includeamino and/or carboxyl terminal fusions as well as intrasequenceinsertions of single or multiple amino acid residues. Insertionsordinarily will be smaller insertions than those of amino or carboxylterminal fusions, for example, on the order of one to four residues.Deletions are characterized by the removal of one or more amino acidresidues from the protein sequence. Typically, no more than about from 2to 6 residues are deleted at any one site within the protein moleculebut deletion can range from 1-30 residues. These variants ordinarily areprepared by site specific mutagenesis of nucleotides in the DNA encodingthe protein, thereby producing DNA encoding the variant, and thereafterexpressing the DNA in recombinant cell culture. Techniques for makingsubstitution mutations at predetermined sites in DNA having a knownsequence are well known and include, for example, M13 primer mutagenesisand PCR mutagenesis Amino acid substitutions are typically of singleresidues, but can occur at a number of different locations at once;insertions usually will be on the order of about from 1 to 10 amino acidresidues. Deletions or insertions preferably are made in adjacent pairs,i.e., a deletion of 2 residues or insertion of 2 residues.Substitutions, deletions, insertions or any combination thereof may becombined to arrive at a final construct. The mutations must not placethe sequence out of reading frame and preferably will not createcomplementary regions that could produce secondary mRNA structure unlesssuch a change in secondary structure of the mRNA is desired.Substitutional variants are those in which at least one residue has beenremoved and a different residue inserted in its place. Suchsubstitutions generally are made in accordance with the following Tables1 are referred to as conservative substitutions.

TABLE 1 Amino Acid Substitutions Original Residue ExemplarySubstitutions Ala Ser Arg Lys Asn Gln Asp Glu Cys Ser Gln Asn Glu AspGly Pro His Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln Met Leu; Ile PheMet; Leu; Tyr Pro Gly Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in Table1, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site or (c) the bulk of the side chain. The substitutions whichin general are expected to produce the greatest changes in the proteinproperties will be those in which (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine, and (e) by increasing the number of sites for sulfationand/or glycosylation.

For example, the replacement of one amino acid residue with another thatis biologically and/or chemically similar is known to those skilled inthe art as a conservative substitution. For example, a conservativesubstitution would be replacing one hydrophobic residue for another, orone polar residue for another. The substitutions include combinationsshown in Table 1. Conservatively substituted variations of eachexplicitly disclosed sequence are included within the polypeptidesprovided herein.

Typically, conservative substitutions have little to no impact on thebiological activity of a resulting polypeptide. In a particular example,a conservative substitution is an amino acid substitution in a peptidethat does not substantially affect the biological function of thepeptide. A peptide can include one or more amino acid substitutions, forexample 2-10 conservative substitutions, 2-5 conservative substitutions,4-9 conservative substitutions, such as 2, 5 or 10 conservativesubstitutions.

A polypeptide can be produced to contain one or more conservativesubstitutions by manipulating the nucleotide sequence that encodes thatpolypeptide using, for example, standard procedures such assite-directed mutagenesis or PCR. Alternatively, a polypeptide can beproduced to contain one or more conservative substitutions by usingstandard peptide synthesis methods. An alanine scan can be used toidentify which amino acid residues in a protein can tolerate an aminoacid substitution. In one example, the biological activity of theprotein is not decreased by more than 25%, for example not more than20%, for example not more than 10%, when an alanine, or otherconservative amino acid (such as those listed below), is substituted forone or more native amino acids.

Further information about conservative substitutions can be found in,among other locations, in Ben-Bassat et al., (J. Bacteriol. 169:751-7,1987), O'Regan et al., (Gene 77:237-51, 1989), Sahin-Toth et al.,(Protein Sci. 3:240-7, 1994), Hochuli et al., (Bio/Technology 6:1321-5,1988) and in standard textbooks of genetics and molecular biology.

Substitutional or deletional mutagenesis can be employed to insert sitesfor N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).Deletions of cysteine or other labile residues also may be desirable.Deletions or substitutions of potential proteolysis sites, e.g. Arg, isaccomplished for example by deleting one of the basic residues orsubstituting one by glutaminyl or histidyl residues.

Certain post-translational derivatizations are the result of the actionof recombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and asparyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Otherpost-translational modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the o-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W. H. Freeman & Co., San Francisco pp 79-86[1983]), acetylation of the N-terminal amine and, in some instances,amidation of the C-terminal carboxyl.

It is understood that there are numerous amino acid and peptide analogswhich can be incorporated into the disclosed compositions. For example,there are numerous D amino acids or amino acids which have a differentfunctional substituent than the amino acids shown in Table 1. Theopposite stereoisomers of naturally occurring peptides are disclosed, aswell as the stereoisomers of peptide analogs. These amino acids canreadily be incorporated into polypeptide chains by charging tRNAmolecules with the amino acid of choice and engineering geneticconstructs that utilize, for example, amber codons, to insert the analogamino acid into a peptide chain in a site specific way (Thorson et al.,Methods in Molec. Biol. 77:43-73 (1991), Zoller, Current Opinion inBiotechnology, 3:348-354 (1992); Ibba, Biotechnology & GeneticEnginerring Reviews 13:197-216 (1995), Cahill et al., TIBS,14(10):400-403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba andHennecke, Bio/technology, 12:678-682 (1994), all of which are hereinincorporated by reference at least for material related to amino acidanalogs).

Molecules can be produced that resemble polypeptides, but which are notconnected via a natural peptide linkage. For example, linkages for aminoacids or amino acid analogs can include CH₂NH—, —CH₂S—,—CH₂—CH₂—CH═CH—(cis and trans), —COCH₂—CH(OH)CH₂—, and —CHH₂SO— (Theseand others can be found in Spatola, A. F. in Chemistry and Biochemistryof Amino Acids, Peptides, and Proteins, B. Weinstein, eds., MarcelDekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983),Vol. 1, Issue 3, Peptide Backbone Modifications (general review);Morley, Trends Pharm Sci (1980) pp. 463-468; Hudson, D. et al., Int JPept Prot Res 14:177-185 (1979) (—CH₂NH—, CH₂CH₂—); Spatola et al. LifeSci 38:1243-1249 (1986) (—CHH₂—S); Hann J. Chem. Soc Perkin Trans.1307-314 (1982) (—CH—CH—, cis and trans); Almquist et al. J. Med. Chem.23:1392-1398 (1980) (—COCH₂—); Jennings-White et al. Tetrahedron Lett23:2533 (1982) (—COCH₂—); Szelke et al. European Appin, EP 45665 CA(1982): 97:39405 (1982) (—CH(OH)CH₂—); Holladay et al. Tetrahedron. Lett24:4401-4404 (1983) (—C(OH)CH₂—); and Hruby Life Sci 31:189-199 (1982)(—CH₂—S—); each of which is incorporated herein by reference. Aparticularly preferred non-peptide linkage is —CH₂NH—. It is understoodthat peptide analogs can have more than one atom between the bond atoms,such as b-alanine, g-aminobutyric acid, and the like.

Amino acid analogs and analogs and peptide analogs often have enhancedor desirable properties, such as, more economical production, greaterchemical stability, enhanced pharmacological properties (half-life,absorption, potency, efficacy, etc.), altered specificity (e.g., abroad-spectrum of biological activities), reduced antigenicity, andothers.

D-amino acids can be used to generate more stable peptides, because Damino acids are not recognized by peptidases and such. Systematicsubstitution of one or more amino acids of a consensus sequence with aD-amino acid of the same type (e.g., D-lysine in place of L-lysine) canbe used to generate more stable peptides. Cysteine residues can be usedto cyclize or attach two or more peptides together. This can bebeneficial to constrain peptides into particular conformations. (Rizoand Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference).

It is understood that one way to define any variants, modifications, orderivatives of the disclosed genes and proteins herein is throughdefining the variants, modification, and derivatives in terms ofhomology to specific known sequences. Specifically disclosed arevariants of the nucleic acids and polypeptides herein disclosed whichhave at 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent homologyto the stated or known sequence. Those of skill in the art readilyunderstand how to determine the homology of two proteins or nucleicacids. For example, the homology can be calculated after aligning thetwo sequences so that the homology is at its highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection. These references are incorporated herein by reference intheir entirety for the methods of calculating homology.

The same types of homology can be obtained for nucleic acids by, forexample, the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment.

The provided compositions may be administered orally, rectally,intracisternally, intraventricular, intracranial, intrathecal,intra-articularly, intravaginally, parenterally (intravenously,intramuscularly, or subcutaneously), locally (powders, ointments, ordrops), by intraperitoneal injection, transdermally, by inhalation or asa buccal or nasal spray. The exact amount of the antibody or therapeuticagent required will vary from subject to subject, depending on the age,weight and general condition of the subject, the severity of the diseasethat is being treated, the location and size of the tumor, theparticular compounds used, the mode of administration, and the like. Anappropriate amount may be determined by one of ordinary skill in the artusing only routine experimentation given the teachings herein. Typicalsingle dosages of antibody range from 0.1-10,000 micrograms, preferablybetween 1 and 100 micrograms. Typical antibody concentrations in acarrier range from 0.2 to 2000 nanograms per delivered milliliter. Forinjection into a joint, volumes of antibody and carrier will varydepending upon the joint, but approximately 0.5-10 ml, and preferably1-5 ml, is injected into a human knee and approximately 0.1-5 ml, andpreferably 1-2 ml into the human ankle.

The composition can further comprise a pharmaceutically acceptablecarrier. By “pharmaceutically acceptable” is meant a material that isnot biologically or otherwise undesirable, which can be administered toan individual along with the selected substrate without causingsignificant undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained.

Depending on the intended mode of administration, the antibody ortherapeutic agent can be in pharmaceutical compositions in the form ofsolid, semi-solid or liquid dosage forms, such as, for example, tablets,suppositories, pills, capsules, powders, liquids, or suspensions,preferably in unit dosage form suitable for single administration of aprecise dosage. The compositions will include an effective amount of theselected substrate in combination with a pharmaceutically acceptablecarrier and, in addition, may include other medicinal agents,pharmaceutical agents, carriers, or diluents. Compositions suitable forparenteral injection may comprise physiologically acceptable sterileaqueous or nonaqueous solutions, dispersions, suspensions or emulsions,and sterile powders for reconstitution into sterile injectable solutionsor dispersions. Examples of suitable aqueous and nonaqueous carriers,diluents, solvents or vehicles include water, ethanol, polyols(propyleneglycol, polyethyleneglycol, glycerol, and the like), suitablemixtures thereof, vegetable oils (such as olive oil) and injectableorganic esters such as ethyl oleate. Proper fluidity can be maintained,for example, by the use of a coating such as lecithin, by themaintenance of the required particle size in the case of dispersions andby the use of surfactants.

These compositions may also contain adjuvants such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. It may also be desirable to include isotonic agents, forexample, sugars, sodium chloride, and the like. Prolonged absorption ofthe injectable pharmaceutical form can be brought about by the use ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is admixed with at least one inert customary excipient (orcarrier) such as sodium citrate or dicalcium phosphate or (a) fillers orextenders, as for example, starches, lactose, sucrose, glucose,mannitol, and silicic acid, (b) binders, as for example,carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone,sucrose, and acacia, (c) humectants, as for example, glycerol, (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain complex silicates, andsodium carbonate, (e) solution retarders, as for example, paraffin, (f)absorption accelerators, as for example, quaternary ammonium compounds,(g) wetting agents, as for example, cetyl alcohol, and glycerolmonostearate, (h) adsorbents, as for example, kaolin and bentonite, and(i) lubricants, as for example, talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, or mixturesthereof. In the case of capsules, tablets, and pills, the dosage formsmay also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethyleneglycols, andthe like.

Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others well known in the art. They may contain opacifyingagents, and can also be of such composition that they release the activecompound or compounds in a certain part of the intestinal tract in adelayed manner. Examples of embedding compositions which can be used arepolymeric substances and waxes. The active compounds can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the active compounds, the liquid dosage forms may containinert diluents commonly used in the art, such as water or othersolvents, solubilizing agents and emulsifiers, as for example, ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl alcohol, benzyl benzoate, propyleneglycol,1,3-butyleneglycol, dimethylformamide, oils, in particular, cottonseedoil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil,glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols and fatty acidesters of sorbitan or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include adjuvants,such as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspendingagents, as for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, or mixtures of thesesubstances, and the like.

Compositions for rectal administrations are preferably suppositorieswhich can be prepared by mixing the compounds of the present inventionwith suitable non-irritating excipients or carriers such as cocoabutter, polyethyleneglycol or a suppository wax, which are solid atordinary temperatures but liquid at body temperature and therefore, meltin the rectum or vaginal cavity and release the active component.

Dosage forms for topical administration of a compound of this inventioninclude ointments, powders, sprays, and inhalants. The active componentis admixed under sterile conditions with a physiologically acceptablecarrier and any preservatives, buffers, or propellants as may berequired. Ophthalmic formulations, ointments, powders, and solutions arealso contemplated as being within the scope of this invention.

The term “pharmaceutically acceptable salts, esters, amides, andprodrugs” as used herein refers to those carboxylate salts, amino acidaddition salts, esters, amides, and prodrugs of the compounds of thepresent invention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of patients without unduetoxicity, irritation, allergic response, and the like, commensurate witha reasonable benefit/risk ratio, and effective for their intended use,as well as the zwitterionic forms, where possible, of the compounds ofthe invention. The term “salts” refers to the relatively non-toxic,inorganic and organic acid addition salts of compounds of the presentinvention. These salts can be prepared in situ during the finalisolation and purification of the compounds or by separately reactingthe purified compound in its free base form with a suitable organic orinorganic acid and isolating the salt thus formed. Representative saltsinclude the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate,acetate, oxalate, valerate, oleate, palmitate, stearate, laurate,borate, benzoate, lactate, phosphate, tosylate, citrate, maleate,fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate,lactobionate, methane sulphonate and laurylsulphonate salts, and thelike. These may include cations based on the alkali and alkaline earthmetals, such as sodium, lithium, potassium, calcium, magnesium, and thelike, as well as non-toxic ammonium, quaternary ammonium and aminecations including, but not limited to ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. (See, for example, S. M. Bargeet al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977, 66:1-19 which isincorporated herein by reference.)

Provided is an isolated nucleic acid comprising double-stranded RNA(dsRNA) for use in RNA interference (RNAi). The dsRNA can be shortinterfering RNA (siRNA) or short hairpin RNA (shRNA). Thus, provided isan isolated nucleic acid comprising an shRNA, wherein the shRNA inhibitsthe expression of a CARD containing protein. The shRNA can be encoded bythe nucleic acid sequence SEQ ID NO:10, 12, 14, or 16.

The disclosed nucleic acids are made up of for example, nucleotides,nucleotide analogs, or nucleotide substitutes. Non-limiting examples ofthese and other molecules are discussed herein. It is understood thatfor example, when a vector is expressed in a cell, the expressed mRNAwill typically be made up of A, C, G, and U. Likewise, it is understoodthat if, for example, an antisense molecule is introduced into a cell orcell environment through for example exogenous delivery, it isadvantagous that the antisense molecule be made up of nucleotide analogsthat reduce the degradation of the antisense molecule in the cellularenvironment.

By “isolated nucleic acid” or “purified nucleic acid” is meant DNA thatis free of the genes that, in the naturally-occurring genome of theorganism from which the DNA of the invention is derived, flank the gene.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector, such as an autonomously replicating plasmidor virus; or incorporated into the genomic DNA of a prokaryote oreukaryote (e.g., a transgene); or which exists as a separate molecule(e.g., a cDNA or a genomic or cDNA fragment produced by PCR, restrictionendonuclease digestion, or chemical or in vitro synthesis). It alsoincludes a recombinant DNA which is part of a hybrid gene encodingadditional polypeptide sequence. The term “isolated nucleic acid” alsorefers to RNA, e.g., an mRNA molecule that is encoded by an isolated DNAmolecule, or that is chemically synthesized, or that is separated orsubstantially free from at least some cellular components, e.g., othertypes of RNA molecules or polypeptide molecules.

Provided herein is a vector comprising any of the nucleic acids providedherein, operably linked to an expression control sequence. Preferredpromoters controlling transcription from vectors in mammalian host cellsmay be obtained from various sources, for example, the genomes ofviruses such as: polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis B virus and most preferably cytomegalovirus, orfrom heterologous mammalian promoters, e.g. beta actin promoter or EF1promoter, or from hybrid or chimeric promoters (e.g., cytomegaloviruspromoter fused to the beta actin promoter). The early and late promotersof the SV40 virus are conveniently obtained as an SV40 restrictionfragment which also contains the SV40 viral origin of replication (Fierset al., Nature, 273: 113 (1978)). The immediate early promoter of thehuman cytomegalovirus is conveniently obtained as a HindIII Erestriction fragment (Greenway, P. J. et al., Gene 18: 355 360 (1982)).Of course, promoters from the host cell or related species also areuseful herein.

“Enhancer” generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′(Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3′(Lusky, M. L., et al., Mol. Cell. Bio. 3: 1108 (1983)) to thetranscription unit. Furthermore, enhancers can be within an intron(Banerji, J. L. et al., Cell 33: 729 (1983)) as well as within thecoding sequence itself (Osborne, T. F., et al., Mol. Cell. Bio. 4: 1293(1984)). They are usually between 10 and 300 bp in length, and theyfunction in cis. Enhancers function to increase transcription fromnearby promoters. Enhancers also often contain response elements thatmediate the regulation of transcription. Promoters can also containresponse elements that mediate the regulation of transcription.Enhancers often determine the regulation of expression of a gene. Whilemany enhancer sequences are now known from mammalian genes (globin,elastase, albumin, fetoprotein and insulin), typically one will use anenhancer from a eukaryotic cell virus for general expression. Preferredexamples are the SV40 enhancer on the late side of the replicationorigin (bp 100 270), the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

The promoter and/or enhancer may be specifically activated either bylight or specific chemical events which trigger their function. Systemscan be regulated by reagents such as tetracycline and dexamethasone,synthetic transcription factors, directed RNA self-cleavage (Yen L. etal. 2004. Nature 431:471-476), and other approaches. There are also waysto enhance viral vector gene expression by exposure to irradiation, suchas gamma irradiation, or alkylating chemotherapy drugs.

The promoter and/or enhancer region can act as a constitutive promoterand/or enhancer to maximize expression of the region of thetranscription unit to be transcribed. In certain constructs the promoterand/or enhancer region be active in all eukaryotic cell types, even ifit is only expressed in a particular type of cell at a particular time.A preferred promoter of this type is the CMV promoter (650 bases). Otherpreferred promoters are SV40 promoters, cytomegalovirus (plus a linkedintron sequence), beta-actin, elongation factor-1 (EF-1) and retroviralvector LTR.

It has been shown that all specific regulatory elements can be clonedand used to construct expression vectors that are selectively expressedin specific cell types such as melanoma cells. The glial fibrillaryacetic protein (GFAP) promoter has been used to selectively expressgenes in cells of glial (astrocytic) origin. The HLA-DR, CD11c, Fascinand CD68 promoters have all been used to selectively express genes inantigen-presenting cells, including macrophages and dendritic cells(Brocker, T., et al. 1997. J Exp Med 185:541-550; Gough P. J. andRaines, E. W. 2003. Blood 101:485-491; Cui, Y. et al. 2002. Blood99:399-408; Sudowe, S. et al. 2003. Mol Ther 8:567-575), and promoterelements from dendritic cell-specific genes (such as CD83) may alsoprove useful in this regard (Berchtold S. et al. 2002. Immunobiology205:231-246).

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells) may also contain sequencesnecessary for the termination of transcription which may affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′ untranslated regions also include transcription termination sites. Itis preferred that the transcription unit also contains a polyadenylationregion. One benefit of this region is that it increases the likelihoodthat the transcribed unit will be processed and transported like mRNA.The identification and use of polyadenylation signals in expressionconstructs is well established. It is preferred that homologouspolyadenylation signals be used in the transgene constructs. In certaintranscription units, the polyadenylation region is derived from the SV40early polyadenylation signal and consists of about 400 bases. It is alsopreferred that the transcribed units contain other standard sequencesalone or in combination with the above sequences improve expressionfrom, or stability of, the construct.

The viral vectors can include nucleic acid sequence encoding a markerproduct. This marker product is used to determine if the gene has beendelivered to the cell and once delivered is being expressed. Preferredmarker genes include the E. coli lacZ gene, which encodes βgalactosidase, green fluorescent protein (GFP), and luciferase.

In some embodiments the marker may be a selectable marker. Examples ofsuitable selectable markers for mammalian cells are dihydrofolatereductase (DHFR), thymidine kinase, neomycin, neomycin analog G418,hygromycin, and puromycin. When such selectable markers are successfullytransferred into a mammalian host cell, the transformed mammalian hostcell can survive if placed under selective pressure. There are twowidely used distinct categories of selective regimes. The first categoryis based on a cell's metabolism and the use of a mutant cell line whichlacks the ability to grow independent of a supplemented media. Twoexamples are: CHO DHFR cells and mouse LTK cells. These cells lack theability to grow without the addition of such nutrients as thymidine orhypoxanthine. Because these cells lack certain genes necessary for acomplete nucleotide synthesis pathway, they cannot survive unless themissing nucleotides are provided in a supplemented media. An alternativeto supplementing the media is to introduce an intact DHFR or TK geneinto cells lacking the respective genes, thus altering their growthrequirements. Individual cells which were not transformed with the DHFRor TK gene will not be capable of survival in non supplemented media.

The second category is dominant selection which refers to a selectionscheme used in any cell type and does not require the use of a mutantcell line. These schemes typically use a drug to arrest growth of a hostcell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, (Southern P. and Berg,P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan,R. C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B.et al., Mol. Cell. Biol. 5: 410 413 (1985)). The three examples employbacterial genes under eukaryotic control to convey resistance to theappropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid)or hygromycin, respectively. Others include the neomycin analog G418 andpuromycin.

Provided is a cell comprising any of the herein provided vectors. Thedisclosed cell can be any cell used to clone or propagate the vectorsprovided herein. Thus, the cell can be from any primary cell culture orestablished cell line. The cell type can be selected by one skilled inthe art based on the choice of vector and desired use.

Provided is an isolated polypeptide comprising the CARD containingprotein binding region of a death receptor, wherein the polypeptidecomprises fewer than 25 amino acid residues.

Thus, the provided polypeptide can be the CARD containing proteinbinding region of TFNR1 (accession no. gi:23312372). The providedpolypeptide can be the CARD containing protein binding region of FasReceptor (accession no. gi:119833). The provided polypeptide can be theCARD containing protein binding region of a TRAIL receptor. Thus, theprovided polypeptide can be the CARD containing protein binding regionof DR4 (accession no. gi:21264525). The provided polypeptide can be theCARD containing protein binding region of DR5 (accession no.gi:3721878). The provided polypeptide can be a fragment of the aminoacid sequence SEQ ID NO:22, wherein the fragment binds a CARD containingprotein disclosed herein. As an illustrative example, the providedpolypeptide can comprise amino acids 250-340 of DR5. Thus, thepolypeptide can comprise the amino acid sequence SEQ ID NO:23. Thepolypeptide can further comprise a fragment of amino acid sequence SEQID NO:23, such that the fragment is capable of binding DDX3. As anexample, the modulator can be a polypeptide comprising amino acids280-310 of DR5. Thus, the modulator can comprise the amino acid sequenceSEQ ID NO:24. As another example, the modulator can be a polypeptidecomprising amino acids 300-330 of DR5. Thus, the modulator can comprisethe amino acid sequence SEQ ID NO:36.

By “binds” is meant that the polypeptide forms non-covalent bonds (e.g.hydrogen bonds) with a CARD containing protein protein with sufficientaffinity that can be detected with standard biochemical methods. In oneaspect, the provided polypeptide binds a CARD containing protein with anaffinity equal to or greater than the death receptor from which it isderived.

The CARD containing protein binding region of the death receptor hasutility as a soluble receptor for competitive inhibition of CARDcontaining protein binding. Thus, provided is a method of blocking CARDcontaining protein binding to a death receptor in a cell, comprisingcontacting the cell with a polypeptide encoding the survival region of adeath receptor, as disclosed herein, or a fragment thereof that blocksthe binding. Also provided is a method of reversing a cell's resistanceto a death receptor agonist in a cell comprising contacting the cellwith the polypeptide.

Provided is an isolated polypeptide comprising the death receptorbinding domain of a CARD containing protein. The provided polypeptidecan be the death receptor binding domain of DDX3 (SEQ ID NO:25,accession no. gi:13514816). The provided polypeptide can be a fragmentof the amino acid sequence SEQ ID NO:25, wherein the fragment binds adeath receptor disclosed herein. DDX3 binds DR5 at approximately aminoacids 200 to 250 and 350 to 400. Thus, the modulator can be apolypeptide comprising amino acids 200 to 250 of DDX3, or fragmentsthereof. Thus, the modulator can comprise the amino acid sequence SEQ IDNO:37. Thus, the modulator can be a polypeptide comprising amino acids350 to 400 of DDX3, or fragments thereof. Thus, the modulator cancomprise the amino acid sequence SEQ ID NO:38. The provided polypeptidecan be the death receptor binding domain of mda-5 (accession no.gi:11344593). The provided polypeptide can be the death receptor bindingdomain of RIG-1 (accession no. gi:6048564).

An isolated polypeptide comprising the death receptor binding domain ofa CARD containing protein has utility as a dominant negative inhibitorof death receptor binding by CARD containing proteins if the polypeptideis unable to inhibit death receptor-induced apoptosis. Thus, in oneaspect, the isolated polypeptide can not bind caspases or IAPs. The CARDmotif responsible for binding IAPs of DDX3 is at approximately aminoacids 50-100. Thus, the provided polypeptide can comprise the deathreceptor binding domain of DDX3 but not comprise amino acids 50-100 ofDDX3. Thus, the modulator can be a polypeptide comprising amino acids200 to 250 and/or amino acids 350 to 400 of DDX3, but not comprisingamino acids 50-100 of DDX3. For example, provided is a polypeptideconsisting of amino acids 151-662 of DDX3. Thus, the modulator cancomprise a polypeptide consisting of the amino acid sequence SEQ IDNO:39.

In a further aspect, the polypeptide can block the association ofendogenous CARD containing proteins with the death receptor. In afurther aspect, the polypeptide can prevent the recruitment of IAPs tothe death receptor. In another aspect, the polypeptide can not inhibitthe recruitment of FADD to death receptor. Any combination of theseaspects is contemplated.

The ability of CARD containing proteins such as DDX3 to inhibit deathreceptor-induced apoptosis is, at least in part, due to the recruitmentof IAPs to the death recptor by the CARD domain of the CARD containingprotein. Thus, provided is a method of blocking the association of IAPswith a death receptor, comprising contacting the cell with the disclosedpolypeptide. Also provided is a method of reversing a cell's resistanceto a death receptor agonist comprising contacting the cell with thedisclosed polypeptide.

The compositions disclosed herein and the compositions necessary toperform the disclosed methods can be made using any method known tothose of skill in the art for that particular reagent or compound unlessotherwise specifically noted.

For example, the nucleic acids can be made using standard chemicalsynthesis methods or can be produced using enzymatic methods or anyother known method. Such methods can range from standard enzymaticdigestion followed by nucleotide fragment isolation (see for example,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989)Chapters 5, 6) to purely synthetic methods, for example, by thecyanoethyl phosphoramidite method using a Milligen or Beckman System1Plus DNA synthesizer (for example, Model 8700 automated synthesizer ofMilligen-Biosearch, Burlington, Mass. or ABI Model 380B). Syntheticmethods useful for making oligonucleotides are also described by Ikutaet al., Ann. Rev. Biochem. 53:323-356 (1984), (phosphotriester andphosphite-triester methods), and Narang et al., Methods Enzymol.,65:610-620 (1980), (phosphotriester method). Protein nucleic acidmolecules can be made using known methods such as those described byNielsen et al., Bioconjug. Chem. 5:3-7 (1994).

One method of producing the disclosed polypeptides is to link two ormore peptides or polypeptides together by protein chemistry techniques.For example, peptides or polypeptides can be chemically synthesizedusing currently available laboratory equipment using either Fmoc(9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl)chemistry. (Applied Biosystems, Inc., Foster City, Calif.). One skilledin the art can readily appreciate that a peptide or polypeptidecorresponding to the disclosed proteins, for example, can be synthesizedby standard chemical reactions. For example, a peptide or polypeptidecan be synthesized and not cleaved from its synthesis resin whereas theother fragment of a peptide or protein can be synthesized andsubsequently cleaved from the resin, thereby exposing a terminal groupwhich is functionally blocked on the other fragment. By peptidecondensation reactions, these two fragments can be covalently joined viaa peptide bond at their carboxyl and amino termini, respectively, toform an antibody, or fragment thereof (Grant G A (1992) SyntheticPeptides: A User Guide. W.H. Freeman and Co., N.Y. (1992); Bodansky Mand Trost B., Ed. (1993) Principles of Peptide Synthesis.Springer-Verlag Inc., NY, which is herein incorporated by reference atleast for material related to peptide synthesis). Alternatively, thepeptide or polypeptide is independently synthesized in vivo as describedherein. Once isolated, these independent peptides or polypeptides may belinked to form a peptide or fragment thereof via similar peptidecondensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segmentsallow relatively short peptide fragments to be joined to produce largerpeptide fragments, polypeptides or whole protein domains (Abrahmsen L etal., Biochemistry, 30:4151 (1991)). Alternatively, native chemicalligation of synthetic peptides can be utilized to syntheticallyconstruct large peptides or polypeptides from shorter peptide fragments.This method consists of a two step chemical reaction (Dawson et al.Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779(1994)). The first step is the chemoselective reaction of an unprotectedsynthetic peptide—thioester with another unprotected peptide segmentcontaining an amino-terminal Cys residue to give a thioester-linkedintermediate as the initial covalent product. Without a change in thereaction conditions, this intermediate undergoes spontaneous, rapidintramolecular reaction to form a native peptide bond at the ligationsite (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I etal., J. Biol. Chem., 269:16075 (1994); Clark-Lewis I et al.,Biochemistry, 30:3128 (1991); Rajarathnam K et al., Biochemistry33:6623-30 (1994)).

Alternatively, unprotected peptide segments are chemically linked wherethe bond formed between the peptide segments as a result of the chemicalligation is an unnatural (non-peptide) bond (Schnolzer, M et al.Science, 256:221 (1992)). This technique has been used to synthesizeanalogs of protein domains as well as large amounts of relatively pureproteins with full biological activity (deLisle Milton R C et al.,Techniques in Protein Chemistry IV. Academic Press, New York, pp.257-267 (1992)).

The following examples are set forth below to illustrate the methods andresults according to the present invention. These examples are notintended to be inclusive of all aspects of the present invention, butrather to illustrate representative methods and results. These examplesare not intended to exclude equivalents and variations of the presentinvention which are apparent to one skilled in the art.

EXAMPLES Example 1 Inducible Resistance of Tumor Cells toTRAIL-R2-Mediated Apoptosis by Generation of a Blockade at the DeathDomain Function

Materials and Methods

Cell Lines, Antibodies, and Reagents:

Human breast cancer cell line, MDA-MB-231, was purchased from theAmerican Tissue Culture Collection (ATCC) (Manassas, Va.). Human ovariancancer cell line, UL-3C, was obtained. Cells were maintained in DMEM orRPMI1640 supplemented with 10% heat-inactivated FCS, 50 μg/mlstreptomycin, and 50 U/mL penicillin (Cellgro, Mediatec, Inc., Herndon,Va.). Anti-human TRAIL-R1 (clone: 2E12) and anti-human TRAIL-R2 (clone:TRA-8) monoclonal antibodies were previously described (Ichikawa, et al.2003; Ichikawa, et al. 2001). Anti-human TRAIL-R2 (clone: 2B4) for flowcytometry and immunoprecipitation assays was developed. Recombinantsoluble TRAIL was purchased from Alexis Biochemicals (San Diego,Calif.). Polyclonal anti-caspase 3 and anti-caspase 8 antibodies werepurchased from BD PharMingen (San Diego, Calif.). Monoclonal anti-humancaspase 2, 3, 8, 9 and 10 antibodies, and monoclonal anti-human Bcl-2,Bcl-xL, Bax, cIAP-1, cIAP-2, XIAP and survivin antibodies were prepared.Polyclonal anti-phospho-SAPK/JNK (Thr¹⁸³/Tyr¹⁸⁵), anti-phospho-p38 MAPK(Thr¹⁸⁰/Tyr¹⁸²), anti-PARP antibodies were purchased from Cell SignalingTechnology, Inc. (Beverly, Mass.). Anti-β-actin antibody was purchasedfrom Sigma (St. Louis, Mo.). Anti-FADD was purchased from TransductionLaboratories (Lexington, Ky.). Anti-FLIP was purchased from ProSci Inc.(Poway, Calif.). All horseradish peroxidase (HRP)-conjugated secondaryreagents were purchased from Southern Biotechnology Associates, Inc.(Birmingham, Ala.).

Flow Cytometry Analysis of Cell Surface Expression of TRAIL-R1 and -R2:

10⁶ cells were incubated with 1 μg/ml biotinylated 2E12 and 1 μg/mlPE-conjugated 2B4 on ice for 30 minutes. After twice wash with FACSbuffer (PBS with 5% FBS and 0.01% NaN₃), cells were incubated withStreptoavidin-Cychrome. 10,000 viable cells were analyzed by FACScanflow cytometer (BD, CA).

Cytotoxicity Analysis of Tumor Cell Susceptibility to TRA-8, 2E12 andTRAIL-Mediated Apoptosis:

Cells (1,000 cells per well) were seeded into 96-well plate intriplicate with eight concentrations (double serial dilutions from 1000ng/ml) of TRA-8, 2E12, or TRAIL. Cell viability was determined afterovernight culture using ATPLITE assay according to the manufacture'sinstructions (Packard Instruments, Meriden, Conn.). The results arepresented as the precentage of viable cells in treated wells compared tomedium control wells.

Induction of Tumor Cell Resistance to TRAIL-R2:

Cells (5×10⁵/ml) were incubated with a starting dose of 1 ng/ml TRA-8for two days. Cells were split with fresh medium and incubated with adouble dose of TRA-8 every two days until TRA-8 dose reached 2,000ng/ml. At each treatment cycle, the cell viability of non-induced(parental) and induced cells treated with an inducing dose of TRA-8 wasdetermined by ATPLITE assay.

Cloning and Sequencing of TRAIL-R2:

The full-length cDNA of TRAIL-R2 were obtained by polymerase chainreaction (PCR) using the platinum DNA proofreading polymerase(Invitrogen). The cDNAs were cloned into pCR2.1-TOPO vector(Invitrogen). At least five independent clones were selected forsequencing.

Western Blot Analysis of Apoptosis-Associated Proteins:

Tumor cells (3×10⁶) were washed twice with cold PBS and lysed with 300μl lysis buffer containing 10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.5 mMEDTA, 1 mM EGTA, 0.1% SDS, 1 mM sodium orthovanadate, and a mixture ofprotease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 μg/mlpepstatin A, 2 μg/ml aprotinin). Lysates were sonicated for 10 seconds,and centrifuged for 20 minutes at 12,000 g. The cell lysates with equalamount of total proteins were boiled for 5 minutes with SDS-PAGE samplebuffer. Total cell lysates were seperated in 8%, 10%, or 12% SDS-PAGE,and electrophoretically transferred to nitrocellulose membrane. Theblots were blocked with 5% nonfat dry milk in TBST buffer (20 mMTris-HCl (pH 7.4), 500 mM NaCl, and 0.1% Tween 20) and incubated withprimary antibody in blocking buffer at 4° C. overnight. The blots werewashed three times with TBST and probed with HRP-conjugated secondaryantibodies for one hour at room temperature. After washing four timeswith TBST, the probed proteins were visualized using the ECL Westernblotting detection system (Amersham Biosciences, Piscataway, N.J.)according to the manufacturer's instructions.

cDNA Array Analysis of Transcriptional Regulation of Apoptosis- and CellSignaling-Associated Genes:

The Human Apoptosis Gene Array (HS-002) and the Human SignalTransduction PathwayFinder Gene Array (HS-008) were purchased fromSuperArray, Inc (Frederick, Md.). Total RNA was extracted from cellsusing the TRIZOL® protocol (Invitrogen, Carlsbad, Calif.). The cDNAprobes were synthesized with ³²P-dCTP. The cDNAs on the membrane blotswere hybridized with the ³²P-dCTP labeled probes at 60° C. overnight.The gene expression profiles were analyzed using the CYCLONEPHOSPHORIMAGERT™ (Packard Instruments, Meridien, Conn.).

Co-Immunoprecipitation of TRAIL-R1 and TRAIL-R2:

10⁷ cells were washed with ice-cold PBS, and lysed for 15 mM on ice withlysis buffer (1% Triton X-100, 150 mM NaCl, 10% glycerol, 20 mM Tris-HCl[pH 7.5], 2 mM EDTA, 0.57 mM PMSF, and a protease inhibitor cocktail).The lysates were then cleared twice by centrifugation at 16,000 g for 10minutes at 4° C. The soluble fraction was incubated with 30 μl TRA-8 or2B4 conjugated Sepharose 4B at 4° C. overnight. After seven washes withlysis buffer and three washes with 10 mM Tris, the bound proteins wereeluted by boiling for 3 minutes in SDS-PAGE loading buffer and separatedin SDS-PAGE. The presence of caspase 8 and FADD was determined byWestern blot analysis.

Two-Dimensional Polyacrylamide Gel Electrophoresis:

After co-immuno-precipitation with 2B4-Sepharose 4B, the proteins wereeluted and desalted with acetone, and reconstituted in the IEF samplebuffer (Bio-Rad, Hercules, Calif.). 160 μg total proteins were loaded inthe IPG strip (Bio-Rad) at room temperature overnight, and thenseparated in the PROTEAN IEF™ Cell (BioRad). The protein strips wereequilibrated with the ReadyPrep Equilibration Buffers (Bio-Rad), andfurther separated in 10% SDS-PAGE gel. Upon completion, the gels werefixed with a buffer containing 10% methanol and 7% acetic acid, andstained with SYPRO™ Ruby Staining buffer (Bio-Rad). The gels were imagedusing the VERSADOC™ Digital Imaging System (Bio-Rad) and analyzed withthe PDQUEST™ software (Bio-Rad).

Results

Induction of Selective Resistance to TRAIL-R2-Mediated Apoptosis.

A Human breast cancer cell line, MDA-231, and a human ovarian cancercell line, UL-3C, were selected for induction of TRAIL-R2 resistancebecause they co-expressed high levels of cell surface TRAIL-R1 and -R2as determined by two-color flow cytometry analysis using anti-TRAIL-R1(2E12) and anti-TRAIL-R2 (2B4) antibodies (FIG. 1A). Two tumor celllines were susceptible to apoptosis induced by agonistic anti-TRAILreceptors antibodies, 2E12 and TRA-8, as well as TRAIL as determined byin vitro cytotoxicity assay (FIG. 1B), indicating that both receptorsfor TRAIL are functional in two tumor cell lines. To determine whetherthese tumor cells develop apoptosis resistance to TRA-8 after treatment,cells were treated starting with a non-apoptosis dose (1 ng/ml) of TRA-8for two days, and the doses were then doubled every two days until 2,000ng/ml before withdraw of TRA-8. Cell viability was measured at each dosein both treated and non-treated cells. At TRA-8 doses lower than 10ng/ml, there was no significant cell death in both treated andnon-treated cells. When TRA-8 doses were increased to 50 ng/ml orhigher, a TRA-8 dose-dependent reduction of cell viability was observedin non-treated cells. In contrast, there was no significant cell deathin treated cells up to 2000 ng/ml (FIG. 1C). These results indicate thatthe repeated treatment of tumor cells with low, non-apoptosis-inducingdoses of TRA-8 induces apoptosis resistance and that the inducedresistance is not due to a selection process by removing apoptosissensitive cells.

Four weeks after withdraw of TRA-8, both parental cells (MDA-231P,UL-3CP) and treated cells (MDA-231R, UL-3CR) were tested for theirsusceptibility to apoptosis induced by TRA-8, 2E12 or TRAIL. Compared tonearly 100% cell death of the parental cells after treatment with 1,000ng/ml TRA-8, no significant cell death was induced in both MDA-231R andUL-3CR cells with a range of concentrations of TRA-8 (FIG. 2A),indicating that the cells become highly resistant to TRA-8-inducedapoptosis. In contrast, the susceptibility of the TRA-8 resistant tumorcells to 2E12-induced apoptosis remained unchanged (FIG. 2B). Althoughthe susceptibility was decreased, the induced TRA-8 resistant cells werestill susceptible to TRAIL-mediated apoptosis (FIG. 2C). These resultsindicate that TRA-8-induced apoptosis resistance is selective forTRAIL-R2. After withdraw of TRA-8, the cells remained a TRA-8 resistantstatus for at least 3 months, and then the susceptibility was slowlyrestored to approximately 30% levels of the parental cells by fourmonths (FIG. 2D), indicating that the induced resistance to TRA-8 waslong lasting but partially reversible.

Induced TRAIL-R2 Resistance is not Due to Altered Cell SurfaceExpression or Mutation of TRAIL-R2 or an Intrinsic Apoptosis Defect.

That TRA-8-induced apoptosis resistance was selective for TRAIL-R2indicates that expression of TRAIL-R2 can be selectively reduced or amutation of TRAIL-R2 can occur after induction of TRA-8 resistance. Torule out these possibilities, cell surface expression of TRAIL-R2 wasexamined, and it was determined that there was no alteration inexpression levels of TRAIL-R2 in both TRA-8 resistant cells compared totheir parental cells (FIG. 3A). This result was further confirmed byWestern blot analysis showing that the two isoforms of TRAIL-R2 proteinwere equally expressed in parental and resistant cells (FIG. 3A). Thefull-length cDNA clones of TRAIL-R2 isolated from both TRA-8 resistantcell lines were sequenced, and no mutations were identified. Theseresults indicated that the induced and selective resistance to TRAIL-R2is not due to alterations of TRAIL-R2 itself.

TRAIL-R2-mediated apoptosis can be regulated by a number of apoptosisregulatory proteins such as the inhibitor of apoptosis (IAP) family(Park, et al. 2002; Ng, et al. 2002; Roa, et al. 2003; Cummins, et al.2004; Li, et al. 2004; Bockbrader, et al. 2005) and the Bcl-2 family(Hinz, et al. 2000; Rokhlin, et al. 2001; Fulda, et al. 2002; Carthy, etal. 2003; Chawla-Sarkar, et al. 2004; Sinicrope, et al. 2004). Using apanel of newly developed monoclonal antibodies, the protein levels ofexpression of cIAP1, cIAP2, XIAP, survivin, Bcl-2, Bcl-xL and Bax wereexamined by Western blot analysis. Although MDA231 and UL-3C expressedvariable levels of these proteins, there was no significant differencebetween the parental and resistant cells (FIG. 3A), indicating that theexpression levels of these proteins are unlikely involved in theinduction of TRA-8. A more broad screening for potential transcriptionalalterations among a panel of apoptosis- and cell signaling-associatedgenes was performed using membrane cDNA arrays (Superarray, Frederick,Md.), which included more than 200 well-known apoptosis-related genes(FIG. 3B, upper panel) and cell signaling genes (FIG. 3B, lower panel).A parallel comparison between MDA231 parental and resistant cellsindicates that there was no significant alteration in the expressionprofile of these genes after induction of TRA-8-resistance.

Selective Blockade of TRAIL-R2 Apoptosis Signal Transduction in TRA-8Resistant Tumor Cells.

Sequential activation of upstream caspase 8 and down-stream caspase 3 isa key event in TRAIL-R2 apoptosis signal transduction. Thus,time-dependent activation of these two caspases was examined. As shownpreviously, the treatment of TRA-8 sensitive parental MDA231 cells withTRA-8 induced activation of caspase 8 (FIG. 4A, upper panel) and caspase3 (FIG. 4A, middle panel) as shown by generation of cleaved fragments ofcaspases after TRA-8 treatment. As a very sensitive marker of caspaseactivation, PARP was quickly cleaved (FIG. 4A, lower panel). However,activation of caspase 8, caspase 3 and subsequent cleavage of PARP didnot occur in the resistant cells after TRA-8 treatment. The failure ofactivation of a caspase cascade is not due to an intrinsic defect incaspase pathways as the 2E12-triggered TRAIL-R1 caspase activationcascade was not impaired in TRA-8 resistant cells (FIG. 4A, left panel).These results indicate that the TRAIL-R2-associated caspase cascade isselectively blocked at the level of the upstream caspase 8 afterinduction of TRA-8 resistance.

Caspase 8-dependent activation of the JNK/p38 kinase pathway plays acritical synergistic role in TRAIL-R2-mediated apoptosis (Ohtsuka, etal. 2003; Ohtsuka, et al. 2002). The activation of the JNK/p38 kinaseswas measured by Western blot analysis of the phosphorylation of JNK/p38kinases during TRA-8 treatment. Correspondent to caspase 8 activation,JNK (FIG. 4B, upper panel) and p38 (FIG. 4B, lower panel) were quicklyphosphorylated in a time-dependent fashion. However, in TRA-8 resistantcells, only 2E12 but not TRA-8 was able to induce phosphorylation of theJNK/p38, indicating that the JNK/p38 kinase pathways are alsoselectively inhibited in TRA-8-resistant cells.

Selective Blockade of TRAIL-R2 Death Domain Function in TRA-8 ResistantTumor Cells.

As FADD and caspase 8 are recruited to the death domain of TRAIL-R2 andare major components of DISC, the capability of forming a DISC atTRAIL-R1 and TRAIL-R2 was examined in both parental and resistant cellsby co-immunoprecipitation assay. In MDA231 parental cells, aftertreatment with TRA-8 or 2E12, there was a time-dependent increase ofFADD (FIG. 5A, upper panel) and caspase 8 (FIG. 5A, middle panel), whichwere co-immunoprecipitated with TRAIL-R2 (FIG. 5A, left panel) orTRAIL-R1 (FIG. 5A, right panel), respectively. In TRA-8 resistant cells,there was no TRAIL-R2 co-immunoprecipited FADD and caspase 8 duringTRA-8-mediated apoptosis, but the co-immunoprecipitation of FADD andcaspase 8 with TRAIL-R1 after 2E12 treatment was not affected.Furthermore, to determine whether cFLIP, an inhibitory competitor forcaspase 8 to the death domain, plays a role in the blockade of DISCformation, the co-immunoprecipitation of cFLIP with TRAIL-R1 andTRAIL-R2 was also examined. In a similar time-dependent pattern, cFLIPwas co-immunoprecipited with TRAIL-R2 during TRA-8-mediated apoptosis inthe parental cells but not in the resistant cells (FIG. 5A, lowerpanel). The co-immunoprecipitation of cFLIP with TRAIL-R1 during2E12-mediated apoptosis was not different between the parental and TRA-8resistant cells. Since there were similar levels of total proteinexpression of FADD, caspase 8 and cFLIP, the failure of the recruitmentof these death domain-associated proteins is not due to defectiveexpression of these proteins. These results indicate that the inducedTRA-8 resistance is likely due to a selective defect for TRAIL-R2 torecruit FADD and caspase 8 in the formation of DISC after TRA-8treatment.

Failure of the assembly of DISC at the death domain of TRAIL-R2 in TRA-8resistant cells indicates that the function and composition of TRAIL-R2protein complex is altered, in which a newly generated or functionallyaltered protein can associate with TRAIL-R2 and prevent the recruitmentof FADD and caspase 8 to the death domain of TRAIL-R2. Thus, theproteomic profiles of TRAIL-R2-associated proteins were compared inTRA-8-sensitive parental and TRA-8-resistant MDA231 cells before andafter TRA-8 treatment by two dimension proteomic and mass spectrometryanalysis. The differentially expressed proteins that wereco-immunoprecipitated with TRAIL-R2 were analyzed by PDQuest software,which led us to focus on three protein spots that were altered duringTRA-8-mediated apoptosis between parental and resistant cells (FIG. 5B).The spot 1 and 2 representing a protein mass with a molecular weight of50 kDa or 20 kDa, respectively, appeared only in MAD-231 parental cellsafter TRA-8 treatment but not in untreated cells and TRA-8-treatedresistant cells, indicating that these proteins are recruited to theTRAIL-R2 during TRA-8-mediated apoptosis. Based on their molecularweight and isoelectric point, the protein in the spot 1 was confirmed ascaspase-8 (FIG. 5C), and the spot 2 as FADD (FIG. 5D by Western blotanalysis. The proteins in the spot 3 were interesting because they wereconstantly associated with TRAIL-R2 and a shift shifted occurred duringTRA-8-mediated apoptosis from a higher molecular weight protein to alower molecular weight protein (FIG. 5E). This conversion appeared to berelevant to the induced TRA-8 resistance as it was only observed inTRA-8-treated MDA231 parental cells but not in resistant cells.Furthermore, mass spectrometry analysis identified both spots werederived from DDX3, a member of the DEAD-box RNA helicase family. Becausea higher molecular weight form of DDX3 is constantly associated withTRAIL-R2 in TRA-8 resistant cells, it can be a factor that prevents therecruitment of FADD and caspase 8 to the death domain of TRAIL-R2.

Reversal of TRA-8 Resistance by Chemotherapeutic Agents.

Chemotherapeutic agents synergistically enhance TRA-8-mediated apoptosisboth in vitro and in vivo (Ohtsuka, et al. 2003; Ohtsuka, et al. 2002;Buchsbaum et al. 2003), particularly in those TRA-8 resistant cells. Todetermine whether chemotherapeutic agents are able to reverse inducedTRA-8 resistance, the effect of a group of chemotherapeutic agents,Adriamycin, Texol, Cisplatin and Bisindolymaleimide VIII (BisVIII), wereexamined on TRA-8-mediated apoptosis of the induced resistant cells. Inthe presence of indicated concentrations of chemotherapeutic agents, aTRA-8 dose-dependent response was restored in both TRA-8 resistantMDA-231 and UL-3C cells (FIG. 6A), indicating that all chemotherapeuticagents are able to reverse TRA-8-induced resistance. Activation of thecaspase cascade in MAD-231 resistant cells after combination treatmentwith Adriamycin and TRA-8 was examined using a panel of monoclonalanti-caspase antibodies. As anti-caspase 8 (clone: 2F4) and anti-caspase2 (clone: 2A3) only recognize the pro-forms of caspase 8 and 2,respectively, the activation of caspase 8 and 2 was demonstrated byreduced amount of the pro-forms due to the cleavage. Anti-caspase 9(Clone: 4B4) and anti-caspase 3 (clone: 1H6) recognize both pro- andcleaved forms of caspase 9 and 3, respectively, their activation wasshown by the presence of the cleaved fragments of caspase 9 and 3. Thesingle agent treatment alone with TRA-8 (FIG. 6B, lane 5) or Adriamycin(FIG. 6B, lane 4) at 4 hours did not induce any significant activationof all tested caspases compared to non-treated controls (FIG. 6B, lane1). In contrast, activation of caspase 2, 9 and 3 was induced as earlyas one hour after combination treatment with Adriamycin and TRA-8 (FIG.6B, lane 2), which was further enhanced at the four hour time point(FIG. 6B, lane 3). Activation of caspase 8 was evident at four-hour timepoint after combination treatment. These results indicate that treatmentwith Ariamycin restored TRAIL-R2-associated caspase cascade in TRA-8resistant cells. In the presence of Ariamycin, TRA-8 was able to triggerthe recruitment of FADD to TRAIL-R2 (FIG. 6C, lane 2 and 3), therecruiting function of TRAIL-R2 was restored by Adriamycin treatment.

Example 2 Role of DDX3 in TRAIL-R2-Mediated Apoptosis

Materials and Methods

Cell Lines, Antibodies, and Reagents.

Human breast cancer cell line, MDA-MB-231, was purchased from theAmerican Tissue Culture Collection (ATCC) (Manassas, Va.). Human ovariancancer cell line, UL-3C, was obtained. Cells were maintained in DMEM orRPMI1640 supplemented with 10% heat-inactivated FCS, 50 ng/mlstreptomycin, and 50 U/mL penicillin (Cellgro, Medi-atec, Inc., Herndon,Va.). Anti-human TRAIL-R1 (clone: 2E12) and anti-human TRAIL-R2 (clone:TRA-8) monoclonal antibodies were previously described (Ichikawa et al.,2003; Ichikawa et al., 2001). Anti-human TRAIL-R2 (clone: 2B4) wasdeveloped for flow cytometry and immunoprecipitation assays. Recombinantsoluble TRAIL was purchased from Alexis Biochemicals (San Diego,Calif.). Polyclonal anti-caspase 3 and anti-caspase 8 antibodies werepurchased from BD Pharmingen (San Diego, Calif.). Monoclonal anti-humancaspase 2, 3, 8, 9 and 10 antibodies, and monoclonal anti-human Bcl-2,Bcl-xL, Bax, cIAP-1, cIAP-2, XIAP, and survivin antibodies, wereprepared. Anti-PARP antibody was purchased from Cell SignalingTechnology, Inc. (Beverly, Mass.). Anti-β-actin antibody was purchasedfrom Sigma. Anti-FADD were purchased from Transduction Laboratories(Lexington, Ky.). All horseradish peroxidase (HRP)-conjugated secondaryreagents were purchased from Southern Biotechnology Associates, Inc.(Birmingham, Ala.). Active Caspase-1, Caspase-2, Caspase-3, Caspase-6,Caspase-7, Caspase-8, Caspase-9, and Caspase-10 were purchased from EMDBiosciences, Inc (San Diego, Calif.). The fluorogenic peptidederivatives Ac-Val-Asp-Val-Asp-AMC (Ac-VDVAD-AMC, 260060M001, SEQ IDNO:40), Ac-Asp-Glu-Val-Asp-amino-4-methylcoumarin (Ac-DEVD-AMC,260031M001, SEQ ID NO:41), andAc-carbonyl-Ile-Glu-Thr-Asp-7-amido-4-methylcoumarin (Z-IETD-AMC,260042M001, SEQ ID NO:42) were purchased from Alexis Biochemicals, SanDiego, Calif. Caspase-2, -3, -8, -10 inhibitor (FMKSP01) were purchasedfrom R&D Systems, Inc.

Cytotoxicity analysis of tumor cell susceptibility to TRA-8, 2E12, andTRAIL-mediated apoptosis. Cells (1,000 cells per well) were seeded into96-well plates in triplicate with eight concentrations (double serialdilutions from 1000 ng/ml) of TRA-8, 2E12, or TRAIL. Cell viability wasdetermined after overnight culture using an ATPLITE™ assay according tothe manufacturer's instructions (Packard Instruments, Meriden, Conn.).The results are presented as the precentage of viable cells in treatedwells compared to medium control wells.

Flow Cytometry.

Cells (10⁶) were washed once with PBS and resuspended in 1 ml cold FACSbuffer (PBS with 5% FBS and 0.01% NaN₃) containing the primary antibody(1 μg/ml of TRA-8). Cells were stained on ice for 60 minutes, thenwashed with 3 ml cold FACS buffer, and incubated with the secondaryantibody (1:100 dilution of PE-conjugated goat anti-mouse IgG) at 4° C.for 60 minutes in the dark. After an additional 3 ml wash with FACSbuffer, 10,000 cells per sample were analyzed by FACSCAN flow cytometer(BD Biosciences, San Jose, Calif.).

Western Blot Analysis of Apoptosis-Associated Proteins.

Tumor cells (3×10⁶) were washed twice with cold PBS and lysed with 300μl lysis buffer containing 10 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.5 mMEDTA, 1 mM EGTA, 0.1% SDS, 1 mM sodium orthovanadate, and a mixture ofprotease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 ng/mlpepstatin A, 2 μg/ml aprotinin). The cell lysates were sonicated for 10seconds and centrifuged for 20 minutes at 12,000 g. The cell lysateswith equal amounts of total proteins were boiled for 5 minutes withSDS-PAGE sample buffer. Total cell lysates were separated in 8%, 10%, or12% SDS-PAGE, and electrophoretically transferred to a nitrocellulosemembrane. The blots were blocked with 5% nonfat dry milk in TBST buffer(20 mM Tris-HCl (pH 7.4), 500 mM NaCl, and 0.1% Tween 20) and incubatedwith primary antibody in blocking buffer at 4° C. overnight. The blotswere washed three times with TBST and probed with HRP-conjugatedsecondary antibodies for 1 hour at room temperature. After being washedfour times with TBST, the probed proteins were visualized using the ECLWestern blotting detection system (Amersham Biosciences) according tothe manufacturer's instructions.

Under-Expression of DDX3.

Design RNAi: An online design tool, BLOOK-iT RNAi Designer (Invitrogen),was used to identify RNAi targets for DDX3. Five targeted shRNAsequences were selected from the top 10 highest scoring RNAi targets(see Table 2).

TABLE 2 shRNA orientation: SENSE-loop-ANTISENSE Construct StrandSequence 1  108- top CACCAAGCTTGCGCTATATTCCTCCTCATTTcgaaAAATSEQ ID NO: 8  128 GAGGAGGAATATAGCGCCTCGAG bottomAAAACTCGAGGCGCTATATTCCTCCTCATTTttcgAAAT SEQ ID NO: 9GAGGAGGAATATAGCGCAAGCTT 2  562- topCACCGGAGAAATTATCATGGGAAACcgaaGTTTCCCAT SEQ ID NO: 10  582 GATAATTTCTCCbottom AAAAGGAGAAATTATCATGGGAAACttcgGTTTCCCAT SEQ ID NO: 11 GATAATTTCTCC3 1554- top CACCGCCAAGTGATATTGAAGAATAaacgTATTCTTCA SEQ ID NO: 12 1574ATATCACTTGGC bottom AAAAGCCAAGTGATATTGAAGAATAcgttTATTCTTCAASEQ ID NO: 13 TATCACTTGGC 4 5′UTR topCACCGCTTTCCAGCGGGTATATTAGcgaaCTAATATACC SEQ ID NO: 14 CGCTGGAAAGC bottomAAAAGCTTTCCAGCGGGTATATTAGttcgCTAATATACC SEQ ID NO: 15 CGCTGGAAAGC 51045- top CACCGCTGATCGGATGTTGGATATGcgaaCATATCCAA SEQ ID NO: 16 1065CATCCGATCAGC bottom AAAAGCTGATCGGATGTTGGATATGttcgCATATCCAACSEQ ID NO: 17 ATCCGATCAGC

They were then cloned into the BLOCK-iT U6 entry vector. The shRNA isdriven by the U6 promoter and can be transiently expressed in mostdividing or nondividing mammalian cell types. Resistant cells weretransfected with RNAi used LIPOFECTAMINE 2000 (Invitrogen) for the RNAiresponse. The decreased DDX3 expression was determined by Western blotanalysis using anti-DDX3 antibody 36 hours after transfection. Oncedecreased DDX3 expression was achieved, the siRNA oligo was synthesized(Target sequence: GGAGAAATTATCATGGGAAAC (SEQ ID NO:27): Sense RNA5′-F1-GGAGAAATTATCATGGGAAAC (F1-SEQ ID NO:27) (F1=fluorescein);Anti-senseRNA 5′-GUUUCCCAUGAUAAUUUCUCC-3′ (SEQ ID NO:28), and RNAicontrol oligo (RI-010-DP) was purchased from Molecula (Columbia, Md.).

Generation of Expression Vectors.

The full-length DDX3 was cloned into pcDNA3.1 plasmid (Invitrogen) witha His tag at the N-terminus of DDX3. DDX3 and TRAIL-R2 cDNA wasgenerated by reverse transcriptase polymerase chain reaction (RT-PCR)performed with total RNA extracted from MDA231 cells using the followingprimer pair: DDX31 forward primer with BamHI:5′-acggatccaaatgagtcatgtggcagtgga-3′ (SEQ ID NO:29); DDX3662 reverseprimer with xhoI-5′-ctctcgagcaaagcaggctcagttaccc-3′ (SEQ ID NO:30).TRAIL-R21 forward primer with KpnI:5′-aaaggtaccagccatggaacaacggggacag-3′ (SEQ ID NO:31); TRAIL-R2441reverse primer with EcoV: 5′-aaagatatcttaggacatggcagagtctgcatt-3′ (SEQID NO:32); the isolated poly-merase chain reaction fragment of DDX3 wasin frame into pcDNA3.1-His vector (Invitrogen). TRAIL-R2 cDNA was clonedinto the pshutter-CMV vector. The correct sequences were confirmed byDNA sequencing.

DDX3/pcDNA3.1-His expression plasmid was generated by deleting the DDX3sequence between the BamHI and xhoI sites. DDX3151 forward primer withBamHI: 5′-acggatccaaatgttttctggaggcaacactggg-3′ (SEQ ID NO:33);TRAIL-R2/pshutter-CMV expression plasmid was generated by deleting theTRAIL-R2 sequence using the following primer: TRAIL-R2340 reverse primerwith EcoRV: 5′-aaagatatcttactgtctcagagtctcagtgggatc-3′ (SEQ ID NO:34);TRAIL-R2330 reverse primer with EcoRV and xhoI:5′-aaagatatcctcgagatttgctggaaccagcagcct-3′ (SEQ ID NO:35).

Constructions of Expression Plasmids for DDX3 in Bacteria.

DDX3 or cIAP1 fragment was inserted into the TOPO100 vector(Invitrogen). The resulting plasmids were transformed into the E. colistrain BL21 (DE3), which was grown in LB media to exponential phases andinduced with 0.4 mM isopropyl-1-thio-β-D-galactopyranoside for 3 hours.Cells were pelleted, resuspended in lysis buffer (30 mM Tris-HCl, pH7.5, 0.1 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 1% Nonidet P-40, and 20 μg/mlPMSF), and sonicated. The supernatant after centrifugation at 14,000×gfor 15 minutes was purified by Ni column. The protein concentration wasdetermined by BCA assay (Pierce, Rockford, Ill.), and ali-quots werestored at 80° C.

Transient Transfections of 293 or 3T3 Cells.

293 or 3T3 cells were transfected with expression vectors usingLIPOFECTAMINET™ 2000 (Invitrogen, Inc.). After 24 hours transfection,protein expression was determined by Western blot analysis usingrespective monoclonal antibody. For co-immunoprecipitation analysis,cells were lysed with immunoprecipitation-lysis buffer containing aprotease inhibitor cocktail.

Co-Immunoprecipitation Assay.

Anti-DDX3 or anti-TRAIL-R2 antibody was conjugated to Sepharose beads(Sigma). The composition of the TRAIL-R2 DISC was determined as follows.5×10⁶ cells (if not otherwise indicated) were treated with 500 ng/ml ofTRA-8 for the indicated time at 37° C. and then lysed inimmunoprecipitation—lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl,0.2% NONIDET P40, and 10% glycerol and complete protease inhibitorcock-tail) or lysed without treatment (unstimulated condition). TheTRAIL-R2 DISC was then precipitated overnight at 4° C. with 30 μl beads.After immunoprecipitation, the beads were washed four times with lysisbuffer. The beads were then washed five times with 10 mM Tris buffer andresuspended in loading buffer for SDS-PAGE and immunoblotting analyses.

Assay of Caspase Activity In Vitro.

Fluorometric assays were conducted in 96-well clear bottom plates, andall measurements were carried out in triplicate wells. 100 μl of assaybuffer (10 mM HEPES pH 7.0, 50 mM NaCl, 2 mM MgCl₂, 5 mM EDTA, and 1 mMDTT) was added. Active caspase-8 and peptide substrates (Ac-IETD-AMC)were added to each well to a final concentration of 100 ng/μl.Co-immunoprecipitation eluted fraction was added to start the reaction.Background fluorescence was measured in wells containing assay buffer,substrate, and lysis buffer without the cell lysates. Assay plates wereincubated at 37° C. for 1 hour. Fluorescence was measured on afluorescence plate reader (Bio-Tek, Winooski, Vt.) set at 355-nmexcitation and 440-nm emission.

In Vitro Caspase Cleavage Assay.

The ability of caspases to cleave DDX3 was examined in an in vitroassay. The cleavage reactions carried out for 30 min at 37° C.,including 10 ul of eluted fraction from TRAIL-R2 co-IP, 10 ul ofreaction buffer (10 mM HEPES [pH 7.0], 50 mM NaCl, 2 mM MgCl₂, 5 mMEGTA, 1 mM DTT, 2 mM ATP), and 5 ul (0.1 U/μl) recombinant active formsof caspases. The cleavage was determined by Western blot with anti-DDX3antibody.

Results

Proteomics Analysis of a Candidate Protein, DDX3, that Causes a Blockadeof the Death Domain of TRAIL-R2 in Resistant Cells.

The spontaneously developed or induced apoptosis resistance to thetherapeutic agents, TRAIL and agonistic antibodies, that target thedeath receptors represents a major obstacle in effective treatment ofcancer with these agents. In order to determine whether alternativecompositions of TRAIL-R2 death domain complexes in resistant cells, theproteomic profiles of existing TRAIL-R2-associated proteins werecompared in TRA-8-sensitive parental and TRA-8-resistant MDA231 cellsbefore and after TRA-8 treatment by two-dimensional proteomic and massspectrometry analysis. In the examination of two-dimensional gelsstained with SYPRO™ ruby (Molecular Probes, Eugene, Oreg.), a proteinspot about ˜80 KDa was found. The association of this protein withTRAIL-R2 block the formation of TRAIL-R2 DISC, thereby causing TRA-8resistance. The ˜80 kd protein was excised from SDS-PAGE and digestedwith trypsin, and peptide sequences were analyzed by mass sepectrometry.The protein amino acid sequences from six digested fragments were 100%identical to the Genbank sequence of human DDX3 (Table 3), indicatingthat DDX3 disassociates from TRAIL-R2 during TRA-8-induced apoptosiscorrelated to DISC formation. If this protein remains associated withthe TRAIL-R2-associated protein complex, it can prevent FADD recruitmentand cause failure of DISC formation.

TABLE 3 DDX3 Fragments Pep- tide Sequence DDX3 SEQ ID 1HVINFDLPSDIEEYVHR aa512-528 SEQ ID NO: 1 2 DFLDEYIFLAVGR aa395-407SEQ ID NO: 2 3 DLLDLLVEAK aa555-564 SEQ ID NO: 3 4 SFLLDLLNATGKaa429-440 SEQ ID NO: 4 5 TAAFLLPILSQIYSDGPGEALR aa231-252 SEQ ID NO: 5 6QYPISLVLAPT aa265-275 SEQ ID NO: 6

DDX3 is a Novel TRAIL-R2-Associated Protein in TRAIL-R2-MediatedApoptosis.

To determine whether DDX3 is indeed associated with TRAIL-R2, thefull-length (aa1-662), N-terminal fragment (aa1-316), and a C-terminalfragment (aa310-662) of DDX3 were cloned into PcDNAIII3.1 with 6-His tagat the N-terminus. These expression vectors were transfected into MDA231parental cells to achieve overexpression of the recombinant full-lengthand deletion mutants of DDX3. However, only full-length DDX3, not itsN-terminal and C-terminal deletion mutants, was associated with TRAIL-R2as detected by co-immunoprecipitation analysis followed by Western blotanalysis using anti-6-His antibody (FIG. 8A). These results confirmedthat DDX3 associated with TRAIL-R2 and its binding to TRAIL-R2 arefull-length dependent. DDX3 was immunoprecipitated with anti-TRAIL-R2 inMDA231 cells.

To further confirm that association of DDX3 with TRAIL-R2, N-terminal,C-terminal fragment, and full-length versions of DDX3 were expressed inE. coli. Proteins were purified and used as an antigen to generatepolyclonal and a panel of monoclonal antibodies against DDX3.TRAIL-R2-associated DDX3 was detected by co-immunoprecipitation andWestern blot analysis using mouse anti-DDX3 monoclonal antibody. Theresults demonstrated that DDX3 was co-immunoprecipitated with TRAIL-R2in both nonapoptotic parental and resistant cells (FIG. 7). There was atime-dependent decrease of DDX3 in TRA-8-sensitive cells but not inTRA-8-resistant cells during apoptosis. In addition, by Western blotanalysis, a rapid decrease and cleavage of TRAIL-R2-associated DDX3during TRA-8-induced apoptosis was observed. This indicated that thecleavage of DDX3 is caspase-dependent. Based on these results, the DDX3sequence was scrutinized for potential cleavage sites at the N-terminal,and a relatively conserved caspase cleavage motif DKSDEDD (SEQ ID NO:46)was found at amino acids 129-135. It is apparent that cleavage occurs onthe DISC and results in a critical functional element of DDX3 beingreleased from TRAIL-R2. The data were compatible with the latter model,which suggests that initiated caspase is rapidly recruited to TRAIL-R2and cleaves DDX3 readily. In addition, FADD and caspase-8 associate withand recruit to TRAIL-R2 to form DISC, which in turn leads to caspasecascade activation correlated to the TRAIL-R2-associated DDX3 cleavage,this indicates that in certain circumstances DDX3 is essential for theapoptotic program, illustrating that DDX3 associates with TRAIL-R2involved TRAIL-R2-mediated apoptosis resistance.

Mapping Interaction Region of DDX3 with TRAIL-R2.

In order to better understand the regulation of DDX3 inTRAIL-R2-mediated signal transduction, the approximate DDX3 region thatis required for binding TRAIL-R2 was determined using HEK293A cells thathad been transiently transfected with plasmids encoding deletion mutantsof DDX3 (FIG. 8A). The interaction of recombinant DDX3 and TRAIL-R2 wasdetermined by co-immunoprecipitation using TRA-8. Full-length DDX3,DDX3Δ201-662, or DDX3Δ1-400 bound to TRAIL-R2. However, neitherDDX3Δ251-662 nor DDX3Δ1-350 could bind to TRAIL-R2 (FIG. 8A), thisindicates that DDX3 has two binding motifs at TRAIL-R2. One is locatedat the N-terminus (aa 200-250); the other is adjacent to aa 350-400.Western blot analysis of lysates from the same cells confirmed theproduction of comparable amounts of wild-type DDX3 and deletionfragments of DDX3, which exclude differences in protein expression as anexplanation for these results.

DDX3 is permanently associated with TRAIL-R2 and correlates with theblockade of FADD recruitment in TRA-8-resistant cells, indicating thatthe TRAIL-R2-associated DDX3 prevents the recruitment of FADD. There canbe a connection between DDX3 and FADD through TRAIL-R2. To test whetherDDX3 and FADD share a common binding motif at the death domain ofTRAIL-R2 or the two binding motifs are close together, so thatpre-engaged DDX3 interferes with the recruitment of FADD, the locationof the DDX3-binding domain in TRAIL-R2 was determined. Vectors encodingthe full-length TRAIL-R2, and a series of amino-terminal domain deletionof TRAIL-R2 including complete deletion of death domain were constructed(FIG. 8B). In an analogous approach to assess the function of DDX3, andto exclude endogenous human TRAIL-R2, a murine fibroblast cell line,NIH3T3, was chosen as the host cell for the co-expression of humanTRAIL-R2 and DDX3. 3T3 cells were co-transfected with plasmids encodingHis-tagged DDX3 and full-length TRAIL-R2, DDX3 and a series of deletionmutants of TRAIL-R2, and DDX3 alone. Cell surface TRAIL-R2 expressionwas examined by flow cytometry using TRA-8 staining. All transfectedcells exhibited similar levels of cell surface TRAIL-R2 (FIG. 8B),indicating that deletion of the intracellular domain did not alter cellsurface TRAIL-R2. In addition, all transfected cells expressed similarlevels of recombinant DDX3, as detected by Western blot analysis oftotal cell lysates using the anti-6-his antibody. The association ofrecombinant DDX3 with the deletion mutants of TRAIL-R2 was examined byco-immunoprecipitation with TRA-8 and Western blot analysis withanti-6-His antibody (FIG. 8B). The interaction of TRAIL-R2 with DDX3 isindependent of the death domain of TRAIL-R2. To further define theTRAIL-R2 binding motif more accurately, further deletion mutants ofTRAIL-R2, D330, and the truncation of TRAIL-R2 (T300-330) wereconstructed (FIG. 8C), co-transfected with DDX3 into 3T3 cells, andanalyzed for their interactions. The results demonstrated that DDX3 didnot bind to the TRAIL-R2 death domain but rather to a membrane proximalregion (aa 300-330) close to the death domain (aa 340-aa 420) (FIG. 8C).This indicates that DDX3 might play a different role from previouslyidenti-fied death domain-associated proteins in TRAIL-R2 signaling. Inaddition, this region is highly homologous with TRAIL-R1 and DcR2 (FIG.8D). These data indicate that DDX3 is a common adaptor proteinassociated with members of the death receptor family.

DDX Contains CARD.

The functional significance of DDX3 in TRAIL-R2-mediated apoptosis wasnext investigated by analyzing the specific property of this molecule.At least two RNA helicases of the DEAD box protein family have beenidentified recently that contain a caspase recruitment domain (CARD).The CARD in these RNA helicases functions as a regulator for apoptosis.As DDX3 plays an important role in the regulation of TRAIL-R2-mediatedapoptosis, DDX3, a member of the helicases of the DEAD box proteinfamily, can have a CARD as well, and the apoptosis inhibitory functionof DDX3 can be directly dependent on the CARD. Thus the possibility thatDDX3 is a CARD protein was examined. Amino acid alignment analysisindicates that DDX3 contains a conserved action motif between aa 50-aa150, as do MDA5 and RIG1. CARD is a homotypic interaction motif. Theproteins containing CARD interact with each other via this domain. AsDDX3 is a novel, highly conserved CARD-containing-helicase, it iscapable of interacting with other CARD proteins. cIAP1, aCARD-containing protein as well, has been widely regarded as aninhibitor of caspase and is recruited to TNFRI and TNFRII to regulateTNTRI-mediated apoptosis. Whether DDX3 is able to interact with cIAP1was tested using anti-DDX3 or anti-TRAIL-R2 antibody in aco-immunoprecipitation experiment. It was determined that cIAP1 can bereadily co-immunoprecipitated with DDX3 and with TRAIL-R2 complexanalyzed by TRAIL-R2 co-immunoprecipitated and DDX3co-immunoprecipitated in both TRA-8 untreated parental and resistantcells (FIG. 8E). However, cIAP1 was rapidly released from theTRAIL-R2-DDX3 complex, and this was correlated to DDX3 cleavage in theparent cells. In contrast, cIAP1 level increased at the TRAIL-R2-DDX3complex in resistant cells after TRA-8 treatment (FIG. 8E). Theseresults indicate that DDX3 could serve as the link between TRAIL-R2 andcIAP1.

Reverse Resistance by Knockdown DDX3.

To study the role of DDX3 in TRAIL-R2 signaling, the importance ofendogenous DDX3 in TRA8-induced apoptosis was examined. As DDX3 did notdecrease in the resistant cells during TRA-8-induced apoptosis, areduced level of expression of DDX3 can be required for cancer cells tobe susceptible to apoptosis. An RNAi strategy was employed to determinethe role of DDX3 in the resistance to TRAIL-R2-mediated apoptosis. Anonline design tool, BLOCK-IT™ RNAi Designer (Invitrogen), was used toidentify RNAi targets for DDX3. Five targeted shRNA sequences wereselected from the top 10 highest scoring RNAi targets and cloned intothe BLOCK-IT™ U6 entry vector. TRA-8-resistant MDA231 cells weretransfected with five RNAi constructs, and protein expression levels ofDDX3 were determined by Western blot analysis using monoclonal anti-DDX3antibody 48 hours post-transfection. Four out of five tested RNAiconstructs were very effective (over 50% reduction) inhibitors of DDX3expression compared to nontransfected or GFP-transfected controls (FIG.9A). The most effective of these constructs, #2, was selected foranalysis of the effect of DDX3 knockdown in TRA-8-mediated apoptosis. Todetermine whether knockdown DDX3 expression reverses TRA-8susceptibility in TRA-8-resistant cells, TRA-8-resistant MDA231 cellswere co-transfected with an RNAi vector (construct #2) and a GFPexpression vector as an indicator of transfected cells. 48 hours aftertransfection, DDX3 was co-immunoprecipitated with TRAIL-R2 and probedwith an anti-DDX3 antibody. As expected, the expression of DDX3significantly decreased compared to the control cells (FIG. 9B).GFP-positive cells were sorted and cultured with various concentrationsof TRA-8 overnight. Using the ATPLITE assay, MDA231 cells transfectedwith GFP and control vectors did not undergo apoptosis after TRA-8treatment, indicating that the cells retained resistance to TRA-8.However, cells co-transfected with the DDX3 RNAi and GFP exhibited TRA-8dose-dependent cell death (FIG. 9C). Using TUNEL staining, a significantnumber of DDX3 knockdown cells were found to be undergoing apoptosis(FIG. 9D). These results indicate that down-regulation of DDX3expression reverses TRA-8 resistance. To further determine the causalrole of DDX3 in TRAIL-R2-mediated apoptosis, DDX3 expression was reducedin a panel of tumor cells and their susceptibility to TRA-8-inducedapoptosis analyzed. DDX3 RNAi reduced the amount of endogenous DDX3 andenhanced the TRA8-induced apoptosis in the panel of tumor cells,including some spontaneous resistant cells (FIG. 9E-F). In contrast,cells transfected with a control oligonucleotide showed normal DDX3expression and remained resistant to TRA-8-induced apoptosis (FIG.9E-F). Thus, DDX3 is a critical component of the TRAIL-R2 signaltransduction apparatus and is essential for resistance toTRAIL-R2-mediated apoptosis.

TRAIL-R2 without DDX3 Binding Region is Pro-Apoptotic.

To test whether, the DDX3 binding motif represents a novel negativeregulatory domain modulating to the death domain function of TRAIL-R2,the apoptotic-inducing function of mutant TRAIL-R2 was compared to thewild-type TRAIL-R2. Cells transfected with TRAIL-R2 without death domainappeared to not respond to TRA-8 treatment, but cells transfected withTRAIL-R2 with a truncated DDX3 binding domain appeared pro-apoptotic andexhibited more susceptibility to TRA-8-induced apoptosis compared towild-type TRAIL-R2-transfected cells (FIG. 10). There was a pronouncedinhibitory effect of DDX3 that could suppress TRAIL-R2-mediatedapoptosis. These findings indicate that DDX3 is an inhibitory mediatorof TRAIL-R2-induced apoptosis.

DDX3 is a CARD Protein Regulating TRAIL-R2-Mediated Apoptosis.

To dissect TRAIL-R2-DDX3-cIAP1 signaling, the region required for itsbinding to cIAP1 was evaluated. As CARD is at the N terminus of DDX3 andis supposed to interact with cIAP1, this region can be responsible forbinding cIAP1. HEK293A cells were transfected with plasmids encodingHis-tagged full-length DDX3, DDX3Δ 51-662, DDX3Δ101-662, DDX3Δ151-662,or DDX3Δ1-350. Both full-length and C-terminal deleted DDX3 were able toco-immunoprecipitate cIAP1, the DDX3 with the first 100 aa deleted wasunable to co-immunoprecipitate cIAP1 (FIG. 11A). These results confirmthat the N-terminal CARD of DDX3 is responsible for recruiting cIAP1 tothe TRAIL-R2 complex. It also indicated that the cIAP1 binding motif islocated at aa 50-100 of DDX3 in front of the cleavage site, aa 129-135(DKSDEDD; SEQ ID NO:46). If DDX3 is cleaved during the TRAIL-R2-mediatedapoptosis, the N-terminal fragment of DDX3 combination with cIAP1 wouldbe disengaged from the TRAIL-R2 complex, thereby relieving theinhibition of cIAP1 to death signaling. Thus, DDX3 is a candidate forcoupling cIAP1 and death receptors to the apoptosis resistance.

To further substantiate this concept, dominant negative mutant DDX3lacking aa 1-150 was used. This mutant DDX3ΔCARD (DDX3Δ151-662) fails tointeract with cIAP1, but is still able to bind to TRAIL-R2 (FIG. 11B).Thus, whether DDX3Δ151-662 could be a dominant negative inhibitor ofendogenous DDX3 by competing with wild-type DDX3 binding TRAIL-R2 wasassessed. Four type cells were transfected with DDX3ΔCARD. As FIG. 5Bshows, DDX3ΔCARD-transfected cells exhibited higher levels of expressionof DDX3ΔCARD compared to endogenous, full-length DDX3, suggesting thatthe truncated DDX3 is able to compete with endogenous DDX3 for TRAIL-R2binding. As FIG. 11B shows, cIAP1 was co-immunoprecipitated with thefull-length DDX3, but not with DDX3ΔCARD, as analyzed by TRAIL-R2-co-IPand Western blotting probed with anti-DDX3 and anti-cIAP1 antibody.Furthermore, the susceptibility of transfected cells to TRA-8-mediatedapoptosis was examined using the ATPLITE assay. Expression of thefull-length recombinant DDX3 did not alter the susceptibility toTRA-8-mediated apoptosis as all tested cells remained resistant afterTRA-8 treatment. However, TRA-8-resistant tumor cells that expressedhigh levels of DDX3ΔCARD regained their susceptibility to TRA-8-inducedapoptosis after down-regulated TRAIL-R2 associated cIAP1. These dataindicate that the inhibition of cIAP1 to TRA-8-induced-apoptosis ismediated by the intact CARD of DDX3. DDX3 lacking the N-terminal CARDmay serve as a dominant negative that partially reverses TRA-8resistance. The potential susceptibility of cancer cells toTRA-8-induced apoptosis could be regulated by the level of DDX3 andcIAP1 on the TRAIL-R2 associated complex.

TRAIL-R2-DDX3-cIAP1 Complex Inhibits Caspase-8 Activation.

DDX3 was quantified to examine how levels of DDX3 present in the cellscorrelated with caspase-8 recruitment and processing at the TRAIL-R2DISC. MDA231 and UL-3C parental and resistant cells were treated withTRA-8 for four hours, and TRAIL-R2 was immunoprecipitated with a newanti-TRAIL-R2 monoclonal antibody (clone: 2B4), which recognizes adifferent TRAIL-R2 epitope than TRA-8. The TRAIL-R2/DDX3/cIAP1 complexwas released from the beads, and the TRAIL-R2-associated DDX3 and cIAP1were subjected to immunoblotting and sandwich ELISA analysis usinganti-DDX3 and anti-cIAP1 antibody. ELISA plates were coated with 2B4anti-TRAIL-R2 antibody to capture the immunoprecipitated TRAIL-R2, andDDX3 and cIAP1 were measured by specific monoclonal antibodies againstDDX3 (3E2) and cIAP1. Treatment of either parental-sensitive orinduced-resistant tumor cells with TRA-8 did not alter TRAIL-R2 proteinlevels (FIG. 12A). However, the TRAIL-R2-associated DDX3 levels weresignificantly altered by TRA-8 treatment in both sensitive and resistantcells. First, untreated resistant cells expressed higher levels ofTRAIL-R2-associated DDX3 compared to untreated sensitive cells asdetected by 3E2 anti-DDX3 antibody (FIG. 12B). Importantly, after TRA-8treatment, the TRAIL-R2-associated DDX3 was significantly increased inTRA-8-resistant cells but demonstrated a marked decrease in sensitivecells. The levels of cIAP1 in TRAIL-R2 complex were also altered in thesame pattern as DDX3 (FIG. 12C). These results suggest that the CARDdomain of DDX3 was released by cleavage from the TRAIL-R2 complex inTRA-8-sensitive cells during apoptosis by the cleavage, whereas DDX3 andcIAP1 indeed were recruited more efficiently to the TRAIL-R2 upon TRA-8stimulation in resistant cells rather than sensitive cells.

To form functional DISC, it is essential for cancer cells to releasecIAP1 from the TRAIL-R2 complex to reduce its suppression to caspaseduring TRA-8-induced apoptosis. This process requires the cleavage ofDDX3, indicating that this step is important to initiating afeed-forward apoptosis amplification loop. Because TRAIL-R2-associatedDDX3 resistance to cleavage is correlated with a failure of DISCformation in resistant cells, DDX3 cleavage susceptibility at theTRAIL-R2-DDX3-cIAP1 complex is different between parental and resistantcells. TRAIL-R2-associated DDX3 cleavage potential by different caspaseswas analyzed in both cells. TRAIL-R2-DDX3-cIAP1 complex wasco-immunoprecipitated with anti-TRAIL-R2 antibody. The eluted fractionfrom the beads was incubated with active caspase-2 and -8. The cleavageof DDX3 was detected by the Western analysis with anti-DDX3 antibody.These results in combination with ELISA analysis (FIG. 12E) demonstratedthat DDX3 cleavage by caspase-8 in resistant cells was highly attenuatedcompared to sensitive cells, although caspase-2 exhibited similarprotease potential in both cells (FIG. 12E). These results indicate thatthere is a functional difference in the DDX3 complex betweenTRA-8-sensitive and -resistant cells. It also indicates that the failureof cleavage of DDX3 by death receptor-associated initial caspases is akey step in the development of TRA-8 resistance.

As cleavage of DDX3 was inhibited in the induced resistant cells, itpromoted a study to determine the step in apoptosis signaling in whichDDX3 inhibits TRAIL-R2-mediated apoptosis. The DDX3/cIAP1 complex waspredicted to inhibit caspase-8 activation; therefore, the activation ofcaspase-8 at the TRAIL-R2-DDX3-cIAP1 complex as one of the firstdetectable events after receptor triggering was examined. To assess theeffect of the TRAIL-R2-DDX3-cIAP1 complex on caspase-8 activation, thecaspase activity was measured using the fluorofenic substrate,Ac-IETD-AMC, incubated with active caspase-8 and DDX3 co-IP elutedfractions from parental sensitive or induced resistant cells. Adose-dependent inhibition of caspase-8 activity was observed over a widerange of dilutions in the TRAIL-R2 co-IP eluted fraction from resistantcells compared to sensitive cells. In addition, purified cIAP1 alsosuppressed caspase-8 protease activity completely (FIG. 12F). It isplausible that DDX3-associated cIAP1 is an inhibitor in the initialactivation of caspase-8, thereby preventing the cleavage of DDX3. Thus,these data provided direct evidence that DDX3-cIAP1 can regulatecaspase-8 activity and indicates that DDX3-cIAP1 is a specific regulatorof caspase-8 engaged by TRAIL-R2.

The effect of TRAIL-R2-DDX3-cIAP1 on caspase-8 activation was examinedby direct analyses of cIAP1-inhibited caspase-8 in combination withcleavage of DDX3 by caspase assay, and showed that DDX3-cIAP1 alsofunctions as a novel type of caspase inhibitor. The DDX3-cIAP1 complexis capable of arresting death receptor pro-apoptotic signals bysuppressing the activation of caspase-8, thereby inhibiting the cleavageof TRAIL-R2-associated DDX3 by the initial caspase. This model showsthat DDX3 protects cells against TRA-8-induced apoptosis via therecruitment of cIAP1 and contributes to the blockage of the deathsignaling pathways in cancer cells.

Example 3 Regulation of DDX3 Binding to DR5 by Serine Phosphorylation ata N-Terminal Region

Bioinformatics search led to identification of a serine-rich domain inDDX3 (SEQ ID NO: 20, corresponding to amino acids 70 to 90 of SEQ IDNO:26) that is conserved for a potential substrate of GSK3 (FIG. 13A).Compared with β-Catenin and glycogen synthetase which are two bestsubstrates of GSK-3, DDX3 has five sequential serines N-terminal to theprimed site. There are several lines of evidence supporting that DDX3 isa substrate for GSK3: (1) DDX3 is directly associated with GSK3α asdemonstrated by co-immunoprecipitation of DDX3 with GSK3α and GSK3 isable to phosphorylate DDX3 (FIG. 13B); (2) GSK3 fails to phosphorylateDDX3 with a point mutation at Ser90 (FIG. 13C). (3) The Ser90 mutantDDX3 exhibits increased disassociation from DR5 and cleavage duringTRA-8-mediated apoptosis (FIG. 13D). These results show that theserine-rich domain at the N-terminal of DDX3 plays a regulatory role inDDX3 association with DR5.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed method and compositions belong. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present method andcompositions, the particularly useful methods, devices, and materialsare as described. Publications cited herein and the material for whichthey are cited are hereby specifically incorporated by reference.Nothing herein is to be construed as an admission that the presentinvention is not entitled to antedate such disclosure by virtue of priorinvention. No admission is made that any reference constitutes priorart. The discussion of references states what their authors assert, andapplicants reserve the right to challenge the accuracy and pertinency ofthe cited documents. It will be clearly understood that, although anumber of publications are referred to herein, such reference does notconstitute an admission that any of these documents forms part of thecommon general knowledge in the art.

Throughout the description and claims of this specification, the word“comprise” and variations of the word, such as “comprising” and“comprises,” means “including but not limited to,” and is not intendedto exclude, for example, other additives, components, integers or steps.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the method and compositions described herein. Suchequivalents are intended to be encompassed by the appended claims.

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What is claimed is:
 1. A method of screening a cell for a biomarker ofresistance to a death receptor agonist comprising (a) selecting a cellthat expresses a death receptor and a modulator of caspase (IAP),wherein the death receptor is DR5 and wherein the IAP is cIAP1 or cIAP2,(b) contacting the cell with a DR5 agonist, and (c) monitoring theassociation of IAP with a CARD containing protein in the cell, whereinthe association of IAPs with the CARD containing protein indicatesresistance to the agonist, wherein the CARD containing protein is apolypeptide having an amino acid sequence with at least 90% homology toDDX3 comprising amino acids 50-100 of SE0 ID NO:26.
 2. The method ofclaim 1, wherein the CARD containing protein is DDX3 comprising aminoacids 50-100 of SEQ ID NO:26.